Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels

ENGINEERED CARBOHYDRATE-BASED MATERIALS FOR BIOMEDICAL APPLICATIONS Polymers, Surfaces, Dendrimers, Nanoparticles, and ...

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ENGINEERED CARBOHYDRATE-BASED MATERIALS FOR BIOMEDICAL APPLICATIONS Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels

Edited by

RAVIN NARAIN University of Alberta Edmonton, Alberta, Canada

A JOHN WILEY & SONS, INC., PUBLICATION

ENGINEERED CARBOHYDRATE-BASED MATERIALS FOR BIOMEDICAL APPLICATIONS

ENGINEERED CARBOHYDRATE-BASED MATERIALS FOR BIOMEDICAL APPLICATIONS Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels

Edited by

RAVIN NARAIN University of Alberta Edmonton, Alberta, Canada

A JOHN WILEY & SONS, INC., PUBLICATION

C 2011 by John Wiley & Sons, Inc. All rights reserved. Copyright

Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com Library of Congress Cataloging-in-Publication Data: Engineered carbohydrate-based materials for biomedical applications : polymers, surfaces, dendrimers, nanoparticles, and hydrogels / edited by Ravin Narain. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-47235-4 (cloth) 1. Carbohydrates–Biotechnology. I. Narain, Ravin. [DNLM: 1. Biopolymers–physiology. 2. Biocompatible Materials. 3. Biomedical Engineering–methods. 4. Dendrimers. 5. Hydrogels. 6. Polysaccharides–chemistry. QT 37.5.P7] TP248.65.P64E54 2011 660.6–dc22 2010039787 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

CONTENTS

PREFACE

vii

CONTRIBUTORS

xi

1 SYNTHESIS OF GLYCOPOLYMERS

1

Samuel Pearson, Gaojian Chen, and Martina H. Stenzel

2 BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

119

Qian Yang

3 CATIONIC GLYCOPOLYMERS

143

Marya Ahmed and Ravin Narain

4 GLYCOPOLYMER BIOCONJUGATES

167

Marya Ahmed and Ravin Narain

5 GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

189

Marya Ahmed and Ravin Narain

6 GLYCONANOPARTICLES: NEW NANOMATERIALS FOR BIOLOGICAL APPLICATIONS

213

Isabel Garc´ıa, Juan Gallo, Marco Marradi, and Soledad Penades ´ v

vi

CONTENTS

7 GLYCODENDRIMERS AND THEIR BIOLOGICAL APPLICATIONS

261

Elizabeth R. Gillies

8 GLYCOSURFACES

307

Anca Mateescu and Maria Vamvakaki

9 CARBOHYDRATE-DERIVED HYDROGELS AND MICROGELS

337

Mitsuhiro Ebara

10

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

355

Archana Bhaw-Luximon

INDEX

397

PREFACE

Carbohydrates are the most abundant, easily accessible and cheap biomolecules in nature. Besides their potential uses as key chemical raw materials and energy production, they have been recognized to play a key role in a wide variety of complex biological processes. They are involved to a large extent in mediating recognition processes through their interactions with proteins and other biological entities. They have been recognized to play a significant role in many important cellular recognition processes including cell growth regulation, differentiation, adhesion, cancer cell metastasis, cellular trafficking, inflammation by bacteria and viruses, and immune response. Individual carbohydrate–protein interactions are generally weak, and multivalent forms of carbohydrate ligands are usually involved in those biological processes. This book has been conceived in order to provide an up-to-date account of the major developments on the biomedical applications of synthetic carbohydrate-based materials. This book is organized into five main themes such as polymers, nanoparticles, surfaces, dendrimers, and hydrogels. Synthetic glycopolymers are essential macromolecules that display many structural and functional features. With functions similar to those of natural carbohydrates, synthetic glycopolymers with specific pendant saccharide moieties can play a significant role in pathological and biological processes via multivalent carbohydrate– protein interactions. With recent progress in organic and polymer chemistry, functional glycopolymers have been prepared with remarkable ease. Carbohydrate-based polymers with different properties were also synthesized, including biodegradable, thermosensitive, and acid-degradable core-crosslinked glyconanoparticles, with neuroactivity and with chiroptical properties. Chapter 1 provides a comprehensive review on the synthesis of glycomonomers and their corresponding glycopolymers via a wide range of organic and polymerization synthesis approach. Some biological vii

viii

PREFACE

interaction studies and applications of glycopolymers, such as in antivirus/bacteria and gene delivery, are also described. Chapter 2 discusses the solution properties of block glycopolymers and their biological relevance. The synthesis of smart block glycopolymers using various polymerization techniques has been discussed. The usage of these smart glycopolymers in tissue engineering, drug delivery, and pathogen interactions is discussed. One of the well-studied types of block glycopolymers is cationic glycopolymers. The use of cationic polymers for gene delivery purposes is a facile technique that is extensively studied as a possible source of noninvasive and efficient gene delivery. Chapter 3 discusses the role and importance of cationic glycopolymers for gene delivery purposes. The brief overview of synthesis of cationic glycopolymers by different polymerization techniques is provided. The detailed study of cationic glycopolymer for gene delivery purposes is specifically discussed. The incorporation of glycopolymers or their corresponding copolymers to macromolecules of choice can further enhance their physiological impact for biological applications. The major challenge in this regard is the synthesis of glycopolymer bioconjugates of controlled dimensions to explore their uses for biomedical applications. Chapter 4 describes the synthetic techniques used in the literature for the production glycopolymer bioconjugates and their importance in biological applications. The facile approaches to synthesize glycopolymer bioconjugates of controlled dimensions are highlighted and their role in biological assays, diagnostics, and in the study of carbohydrate- and protein-based interactions is elaborated. The synthesis of glycoclusters is an important aspect of synthetic carbohydratebased materials under study to understand their interactions with macromolecules such as pathogens and several proteins. These interactions of glycoclusters with living organisms or macromolecules make the basics of most biological phenomena, including invasion, metastasis, and infections. Nanotechnology is a rapidly growing field of materials science that has also extensively been explored in biological applications, owing to the facile introduction of functional groups on the surface of nanomaterials. The introduction of glycopolymer-based moieties on the surface of nanomaterials are found to produce glycoclusters with enhanced biological significance compared to glycopolymers alone, due to the multivalent effect of functional groups present on the surface of nanomaterials. These nanomaterials are largely studied in literature as a function of their structure, nature of materials, surface functionalization properties, and morphology-dependent interactions with living organisms. Chapter 5 discusses the various strategies to synthesize glycopolymer-functionalized carbon nanotubes and their interactions in vitro and in vivo. The inherent properties of carbon nanotubes toward cellular uptake and their toxicity issues are discussed. Moreover, the uses of glycopolymer-functionalized nanotubes for biomedical applications, including gene and drug delivery, and tissue engineering is described. Chapter 6 provides a brief overview about the synthesis and surface functionalization of another type of nanomaterial, namely metallic nanoparticles. The synthesis and surface functionalization of gold and magnetic nanoparticles and of quantum dots with biocompatible carbohydrate-based polymers has opened various possibilities for their uses in biotechnology and biomedicines. This chapter describes a review of few biomedical applications of these glyconanoparticles, including their use in pathogen

PREFACE

ix

inhibition, fluorescent probes, magnetic resonance imaging, and cancer metastasis. Another approach to obtain multivalency and to enhance the function of glycopolymers is the synthesis of glycodendrimers, which compared to their corresponding polymers are of controlled molecular weight and architecture. Chapter 7 describes the synthesis of glycodendrimers using various strategies and their interactions with proteins are studied. The interactions of glycodendrimers with various proteins at physiological and pathological levels are the discussed. In addition to the surface functionalization of nanoscaffolds in colloidal form, the synthesis of glycopolymer-coated macroscaffolds are found to be an attractive platform for the tissue engineering purposes. These glycopolymer-modified surfaces provide not only biocompatibility but are also shown to possess the potential to provide the selectivity in cellular growth and proliferation. Chapter 8 provides a detailed overview of the synthetic techniques involved in the functionalization of macroscaffolds with glycopolymers or their corresponding copolymers. Moreover, the characterization of these surfaces and their role in tissue engineering and as nonfouling surfaces for the inhibition of pathogens is discussed. Chapter 9 provides a different synthetic route to produce biomaterials for tissue engineering and gene delivery. The chapter focuses on the synthesis of glycopolymer-functionalized hydrogels by various techniques. The use of these hydrogels in tissue engineering and drug delivery is discussed. Chapter 10 mainly focuses on the modification of natural carbohydrate-based scaffolds for drug delivery purposes via various administration routes. These modifications are thought to increase the efficacy of drug delivery, in addition to eliminating the hypersensitivity reactions associated with various drug treatments. Ravin Narain

CONTRIBUTORS

Marya Ahmed, Department of Chemical and Materials Engineering and Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, AB, Canada Archana Bhaw-Luximon, Department of Chemistry, University of Mauritius, R´eduit, Mauritius Gaojian Chen, Centre for Advanced Macromolecular Design, University of New South Wales, Sydney, Australia Mitsuhiro Ebara, Smart Biomaterials Group, Biomaterials Center, National Institute for Materials Science, Tsukuba, Japan Juan Gallo, Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE/CIBER-BBN, San Sebastian, Spain Isabel Garc´ıa, Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE/CIBER-BBN, San Sebastian, Spain Elizabeth R. Gillies, Department of Chemistry, Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Canada Marco Marradi, Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE/CIBER-BBN, San Sebastian, Spain Anca Mateescu, Institute of Electronic Structure and Laser, Foundation for Research and Technology—Hellas Heraklion, Crete, Greece, Department of Chemistry, University of Crete, Heraklion, Crete, Greece

xi

xii

CONTRIBUTORS

Ravin Narain, Department of Chemical and Materials Engineering and Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, AB, Canada Samuel Pearson, Centre for Advanced Macromolecular Design, University of New South Wales, Sydney, Australia Soledad Penad´es, Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE/CIBER-BBN, San Sebastian, Spain Martina H. Stenzel, Centre for Advanced Macromolecular Design, University of New South Wales, Sydney, Australia Maria Vamvakaki, Institute of Electronic Structure and Laser, Foundation for Research and Technology—Hellas, Heraklion, Crete, Greece, Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece Qian Yang, Lehrstuhl f¨ur Technische Chemie II, Universit¨at Duisburg-Essen, Essen, Germany

CHAPTER 1

SYNTHESIS OF GLYCOPOLYMERS SAMUEL PEARSON, GAOJIAN CHEN, and MARTINA H. STENZEL Centre for Advanced Macromolecular Design, University of New South Wales, Sydney, Australia

1.1 Introduction 1.2 Synthesis of Vinyl-Containing Glycomonomers 1.2.1 Monomers from Protected Carbohydrates 1.2.2 Monomers from Unprotected Sugars 1.3 Conventional Free Radical Polymerization 1.3.1 Acrylamide Monomers 1.3.2 (Meth)acrylate Monomers 1.3.3 Styrene-Based Monomers 1.3.4 Other Vinyl-Containing Glycomonomers 1.4 Controlled/Living Radical Polymerization 1.4.1 Stable Free Radical Polymerization 1.4.2 Atom Transfer Radical Polymerization 1.4.3 Reversible Addition–Fragmentation Chain Transfer Polymerization 1.5 Ring-Opening Polymerization 1.6 Ionic Chain Polymerization 1.6.1 Anionic Chain Polymerization 1.6.2 Cationic Chain Polymerization 1.7 Ring-Opening Metathesis Polymerization (ROMP) 1.8 Postfunctionalization of Preformed Polymers Using Sugar Moieties 1.8.1 Amide Linkage 1.8.2 Click Approach 1.8.3 Other Nonclick Approaches 1.9 Conclusions References

2 2 2 4 5 6 6 18 19 20 20 33 50 71 74 74 80 82 90 98 101 103 104 104

Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

1

2

SYNTHESIS OF GLYCOPOLYMERS

1.1 INTRODUCTION Glycopolymers—synthetic polymers with pendant carbohydrates—have received considerable attention in the fields of polymer chemistry, material science, and biomedicine due to their biocompatibility and their bioactivity. From humble beginnings where glycopolymers were synthesized from vinyl-functionalized sugars via free radical polymerization with little control over the resulting polymer characteristics, glycopolymer synthesis has now developed into a mature area where the control over molecular weight and polymer architecture is routinely sought and indeed achieved. Glycopolymer synthesis has now infiltrated most known techniques of polymer synthesis and is not restricted to controlled radical processes; ionic techniques also provide feasible means to polymerize glycomonomers in a controlled manner. The aim of this chapter is to provide a comprehensive review of all the techniques that have been utilized for the synthesis of glycopolymers; however, greater emphasis will be placed on the techniques that supercede free radical polymerization. After highlighting the important achievements in the synthesis of glycopolymers via free radical polymerization, focus will turn toward glycopolymer synthesis via the controlled/living radical polymerization processes known as nitroxide-mediated polymerization (NMP), cyanoxyl-mediated polymerization, atom transfer radical polymerization (ATRP), and reversible addition–fragmentation chain transfer (RAFT) polymerization. Glycopolymer synthesis by ring-opening polymerization (ROP), anionic polymerization and cationic polymerization will detail the progress made in the area of ionic polymerization, and a discussion of the work carried out using ring-opening metathesis polymerization (ROMP) will conclude the section on the synthesis of glycopolymers by polymerizing sugar-containing monomers. The functionalization of reactive polymer scaffolds with carbohydrate species will then be discussed as an alternative strategy for synthesizing glycopolymers.

1.2 SYNTHESIS OF VINYL-CONTAINING GLYCOMONOMERS 1.2.1 Monomers from Protected Carbohydrates The commercial availability of a range of carbohydrates provides access to a wide array of different glycomonomers, and significant efforts have been dedicated to the synthesis of polymerizable vinyl sugars. In an early feature article, Wulff et al. [1] highlighted possible avenues for generating glycomonomers in which an important distinction must be made between protected and unprotected carbohydrates. The choice of employing protected or unprotected sugars is dependent on the ease of stereospecific functionalization of the sugar, the solubility of the monomer and polymer, the potential incompleteness of the removal of the protective group, and the ease of purification. The most common synthetic approaches are outlined below, but they are discussed in more detail elsewhere [1].

SYNTHESIS OF VINYL-CONTAINING GLYCOMONOMERS

3

1.2.1.1 Reactions Using Isopropylidene-Protected Sugars Many sugars can easily be protected using acetone to form isopropylidene derivatives. This approach, which is suitable for a range of sugars including glucose, galactose, fructose, and sorbose, allows easy functionalization of the remaining hydroxyl functionality with acrylate [2], methacrylate [3], and 4-vinylbenzyl groups [4].

1.2.1.2 Glycosides from Halogeno Sugars Glycoside monomer synthesis via the reaction between halogeno sugars and hydroxyl groups of vinyl-containing species has been explored in detailed with varying degrees of success. The starting materials, typically acetylated 1-halogeno sugars, can be expensive or difficult to obtain, but the technique is especially useful for inserting longer spacers between the polymerizable moiety and the sugar. The cleavage of the acetyl protecting groups in alkaline media does not affect the glycoside bond [5].

1.2.1.3 Grignard Reactions The aldehyde functionality of a sugar molecule can be targeted by Grignard reagents [6]. Prior protection of the remaining hydroxyl groups is essential.

4

SYNTHESIS OF GLYCOPOLYMERS

1.2.2 Monomers from Unprotected Sugars

1.2.2.1 Enzymatic trans-esterification Enzyme-catalysed transesterification reactions present a highly efficient and regioselective avenue for obtaining glycomonomers that would otherwise be inaccessible without utilizing protecting groups. The reaction of sugars with vinyl-containing esters in organic solvents is catalyzed by lipases such as Candida antarctica, usually yielding derivatives functionalized in 6-position [7], although efficient functionalization in the 1-position has also been reported [8].

1.2.2.2 Fischer Glycoside Synthesis Direct monosubstitution in the anomeric (C-1) position without the recourse to protective chemistry can be achieved by the reaction of an excess of hydroxyl groups, such as in hydroxyethyl acrylate, with the sugar in the presence of phosphomolybdic acid as catalyst [9].

β

1.2.2.3 Synthesis via Barbituratic Acid Barbituratic acid reacts readily with the C-1 position of the unprotected sugar to generate a reactive salt. Subsequent reaction with bromides such as 4-vinyl benzyl bromide leads to polymerizable monomers. Conversion of the barbituratic acid ring to a diamide further improves water solubility [10].

1.2.2.4 Conversion of Aminosugars A popular route to glycomonomers is the fast reaction between aminosugars and acyl halides or anhydrides. The high reactivity of the amine group ensures its preferential reaction even in the presence of unprotected hydroxyl groups. Reactions of acryloyl chloride and methacryloyl

CONVENTIONAL FREE RADICAL POLYMERIZATION

5

chloride [11] and also isocyanates [12] and epoxides with various aminosugars have been explored to confer the glycomonomers in high yields.

1.2.2.5 Reaction between Oxidized Sugars and Amines A range of amide-linked glycomonomers are accessible from sugars that have been oxidized to their corresponding lactones and can therefore be reacted with vinyl-functionalized amines [13].

While these are the most common strategies used for glycomonomer synthesis, other pathways have emerged in recent years such as Cu(I) click chemistry [14]. Some of these approaches are highlighted in this chapter.

1.3 CONVENTIONAL FREE RADICAL POLYMERIZATION Free radical polymerization is one of the most widely used techniques for making polymers. The polymerization reaction is initiated by free radical initiators and has been used to synthesize linear vinyl saccharide polymers since the 1960s. Despite its disadvantages, such as high polydispersities of the resulting polymers and difficulties in controlling terminal functionalities, the robustness of free radical polymerization has encouraged its widespread use. Indeed, a large number of reports have emerged on the synthesis of glycopolymers via free radical polymerization in both aqueous and nonaqueous media. Glycopolymers, polymers with pendant sugar groups, were first reported in 1961 when Kimura et al. [15] and Whistler et al. [11a, 11c] reported the free radical homo- and copolymerization of glycomonomers. Significant activity in the field of free radical polymerization of glycomonomers emerged in the following years, which only declined in the late 1990s with the birth of living free radical polymerization

6

SYNTHESIS OF GLYCOPOLYMERS

techniques. The feature article by Wulff et al. [1] highlighted the body of work and the array of structures. Here, we only highlight some of the latest achievements in this area, mainly publications after 1990. 1.3.1 Acrylamide Monomers Roy et al. copolymerized 4-acrylamidophenyl-␤-lactoside (Table 1.1, entry 1) and acrylamide in water at 90◦ C in the presence of ammonium persulfate (APS) and tetramethylethylenediamine (TMEDA) [16]. The antigenicity of the resulting carbohydrate copolymer was then demonstrated by agar gel diffusion with peanut and castor bean lectins. Nishimura et al. outlined the synthesis of 3-(N-acryloylamino)propyl 2-acetamido-2-deoxy-␤-d-glucopyranoside [17] (Table 1.1, entry 2) and 3-(N-acryloylamino)propyl O-(␤-d-galactopyranosyl)-(l-4)-2acetamido-2-deoxy-␤-d-glucopyranoside [18] (Table 1.1, entry 3) and polymerized them in a similar manner to Roy et al. [16]. Methacrylamide-functionalized mannose monomers (Table 1.1, entries 4–5) were polymerized by Tagawa et al. using a lipophilic azo-initiator containing two long alkyl chains per initiating fragment [19]. Incorporation of the amphiphiles into liposomes generated structures that were able to recognize Concanavalin A (Con A) with little difference observed between the species with varying spacer lengths between the polymer backbone and the sugar residue. Also starting with protected sugars, Carpino et al. reported the synthesis of 2,3,4,6-tetra-O-acetyl-1-O-(4methacryloylaminophenyl)-␤-d-glucopyranoside (Table 1.1, entry 6) and 1-O-(4methacryloylaminophenyl)-␤-d-glucopyranoside (Table 1.1, entry 7), and homopolymerized them with 2,2 -azobisisobutyronitrile (AIBN) as initiator in dimethylformamide (DMF) to afford polymers that were then deprotected with sodium methoxide to give water-soluble glycopolymers [20]. 1.3.2 (Meth)acrylate Monomers Novel (meth)acrylic monomers (Table 1.1, entry 9) bearing a monosaccharide residue were developed by Kitazawa et al. by reacting methyl glycosides with 2-hydroxyethyl acrylate or methacrylate in the presence of heteropoly acid. The monomers were then polymerized in aqueous solution with potassium persulfate as initiator [9]. A galactose-based monomer containing a galactopyranose unit attached through an ester linkage to a vinyl group (Table 1.1, entry 10) was synthesized by Fortes and co-workers and was then copolymerized with ethyl acrylate in DMF under free radical conditions [22]. The protected monomers 2-(2 ,3 ,4 ,6 -tetra-O-acetyl-␤-d-glucosyloxy)ethyl methacrylate (Table 1.1, entry 11) and 2-(2 ,3 ,4 ,6 -tetra-O-acetyl-␤-dgalactosyloxy)ethyl methacrylate (Table 1.1, entry 12) were polymerized by Cameron et al. in chloroform, and the polymers deacetylated in a mixture of dichloromethane and methanol [23]. The alternative approach for obtaining deprotected polymers was also adopted; entries 11 and 12 were deprotected to give the corresponding monomers 2-(␤-d-glucosyloxy)ethyl methacrylate (GlcEMA; Table 1.1, entry 13) and

7

1

2

3

4

Glucosamine

Lactosamine

Mannose

HO

HO

O

OH OH

O

OH

4

OH

O

S

O

O

HO

NHAc

O

n = 3 or 6 HO OH

HO HO

OH

Entry Monomer

2

O

NHAc

O

H N O

n = 3 or 6

OH

H N n

O n

H N O

Glycomonomers Synthesized via Free Radical Polymerization

Lactose

Carbohydrate

TABLE 1.1

2:1 THF/MeOH

H2 O/DMSO

H2 O

H2 O

Solventa

70◦ C

DODA-ACPA

APS

APS

25◦ C

50◦ C

APS

90 C

◦

Temperature Initiatorb

(continued)

19

18

17

16

Reference

8 5

6

7

Glucose

Glucose

O

n = 4 or 7

HO

OH OH

O

OH

Entry Monomer

Mannose

Carbohydrate

n

H N O

TABLE 1.1 Glycomonomers Synthesized via Free Radical Polymerization (Continued)

DMF

DMF

n = 4: H2 O n = 7: 1:1 THF/MeOH

Solventa

AIBN

AIBN

60◦ C

V50 DODA-ACPA

60◦ C

70◦ C 70◦ C

Temperature Initiatorb

20

20

19

Reference

9

8

9

10

11

Glucose

Glucose Galactose Mannose Xylose

Galactose

Glucose

Chloroform

DMF

H2 O

Benzene

AIBN

AIBN

80◦ C

65◦ C

No mention KPS in the paper

40 or 50◦ C AIBN

(continued)

23a

22

9

21

10 12

13

14

15

16

Glucose

Galactose

Glucose

Galactose AcO

AcO OAc

HO

HO O H

AcO

AcO OAc

Entry Monomer

Galactose

Carbohydrate

AcO

O

OH

O

AcO

O

O

O

O

O

CO2Et

O

O

O

TABLE 1.1 Glycomonomers Synthesized via Free Radical Polymerization (Continued)

Chlorobenzene

Chlorobenzene

H2 O/MeOH

H2 O/MeOH

Chloroform

Solventa

AIBN

AIBN

70◦ C

K2 S2 O8

65◦ C

70◦ C

K2 S2 O8

AIBN

65◦ C

65 C

◦

Temperature Initiatorb

24

24

23a, 23b

23a

23a, 23b

Reference

11

17

18

19

Arabinose

Fructose

Galactose

HO

OH

OH

OH

OH

OH

OH,H

OH OH

O

OH,H

HO

OH

HO

HO

HO

OH, H

H2 O

H2 O

H2 O, DMF

70◦ C

70◦ C

70◦ C

AAPD

AAPD

(continued)

25

25

AAPD, AIBN 6b-d, 25, 26

12 20

21

22

Lactose

Maltose

HO

HO OH

Entry Monomer

Lactose

Carbohydrate

OH

O O HO

OH

N R

O

R = H or CH3

HO

OH N H

TABLE 1.1 Glycomonomers Synthesized via Free Radical Polymerization (Continued)

DMSO, H2 O

H2 O

H2 O

Solventa

60◦ C

25◦ C

RT

c

AIBN, KPS

KPS

KPS

Temperature Initiatorb

13b, 30

28

27

Reference

13

23

24

25

26

Lactose

Maltotriose

Lactose

Lactosamine HO

HO OH

HO

HO OH

AcHN

O

OH

O

O

O

HO

HO

OH

OH

AcHN

O

OH

O

H N

H N

O

O

DMSO

DMSO

DMSO, H2 O

DMSO, H2 O

AIBN

60◦ C

AIBN

AIBN, KPS

60◦ C

60◦ C

AIBN, KPS

60◦ C

(continued)

29

29

13b

13b, 30

14 27

28

29

Glucuronamide

Galactose/ gluconamide HO

HO OH

HO

O

HO HO

Entry Monomer

Glucose

Carbohydrate

O

OH

OH O

NH

TABLE 1.1 Glycomonomers Synthesized via Free Radical Polymerization (Continued)

DMSO

DMSO

DMSO

Solventa AIBN

AIBN

AIBN

60◦ C

60◦ C

60◦ C

Temperature Initiatorb

30

30

30

Reference

15

30

31

32

␣,␣-Galactotrehalose

␣,␤-Galactotrehalose

Glucosamine HO HO

HO HO

HO HO

O

O

H N

n

OH

NH

O

O

OH

NH

O

O

OH

O HN

OH

NHAc

O

O

HN

HN

n = 1 or 2 or 3 or 9

OH

HO O HO

O

OH

HO

HO O

O

OH

O

H2 O

DMSO, H2 O

DMSO, H2 O

RT

60◦ C

60◦ C

APS

AAPD

AAPD

(continued)

32

31

31

16 33

34

35

36

Chitobiose

Lactosamine

Galactose O O

HO

HO

Me

HO OH

HO HO

OH

Entry Monomer

Lactosamine

Carbohydrate

O

O O O

HO

OH

O

O

AcHN

O

O

OH

OH

HO

O

O

OH

NHAc

O

AcHN

O

O

O

TABLE 1.1 Glycomonomers Synthesized via Free Radical Polymerization (Continued)

Copolymerization in different solvents

H2 O

H2 O

H2 O

Solventa

65◦ C

RT

RT

RT

AIBN

APS

APS

APS

Temperature Initiatorb

34

33

32

32

Reference

17

38

39

Lactitol

Glucose

HO

HO

HO HO

HO

O

OH O HO

HO O HO

O

OH

O

OH

O

OH OH

O

O

OH

OH OH

O

(CH2)8

O

O (CH2)8

O

H2 O or methanol ethanol 2-propanol

H2 O/DMSO

H2 O/DMSO

60◦ C

60◦ C or 35◦ C

60◦ C

ACPA

AAPD or H2 O2 with l-ascorbic acid

AAPD or H2 O2 with l-ascorbic acid

b APS

a DMSO

= dimethyl sulfoxide; THF = tetrahydrofuran; MeOH = methanol; DMF = N,N-dimethyl formamide. = ammonium persulfate; DODA-ACPA = dioctadecylamine-functionalised 4,4 -azobis(cyanopentanoic acid); V50 = 2,2 -azobis(2-methylpropionamidine) dihydrochloride; AIBN = 2,2 -azobisisobutyronitrile; KPS = potassium persulfate; AAPD = 2,2 -azobis(2-amidinopropane)dihydrochloride. c RT = room temperature.

37

Maltitol

36

35

35

18

SYNTHESIS OF GLYCOPOLYMERS

2-(␤-d-galactosyloxy)ethyl methacrylate (GalEMA; Table 1.1, entry 14), which were then polymerized in a water–methanol mixture. Poly(GalEMA) synthesized via the second approach was tested for the binding with peanut agglutinin (PNA) and the thermodynamic binding parameters were calculated [23]. In another report, Cuervo-Rodriguez and co-workers synthesized methacrylate derivatives bearing acetylated glucopyranoside (Table 1.1, entry 15) and galactopyranoside (Table 1.1, entry 16) residues. Glycopolymers were then obtained by homopolymerization (and copolymerization with methyl methacrylate) in chlorobenzene, and their binding to Ricinus communis agglutinin (RCA120 ) was investigated after deprotection using methoxide [24]. 1.3.3 Styrene-Based Monomers Wulff et al. invested significant effort into the synthesis and polymerization of styrenic glycomonomers (Table 1.1, entries 17–19). The oxidation of sugars to aldehydes and a subsequent Grignard reaction using 4-vinyl-phenylmagnesium chloride was the preferred method for generating the glycomonomers [6b]. Deprotection after the free radical polymerization in water produced polymers with high molecular weights [25]. Narain et al. polymerized the same monomer 4vinylphenyl-d-gluco(d-manno)hexitol (Table 1.1, entry 17) using 2,2 -azobis-(2amidinopropan)dihydrochloride (AAPD) initiator in water to obtain copolymers with acrylamide [26]. Thermal properties of the polymers were studied by differential scanning calorimetry (DSC). In the same report, the monomer was copolymerized with styrene in DMF using AIBN as initiator. Kurth and co-workers prepared a new type of sugar monomer with an oxime linkage, d-lactose-O-(p-vinylbenzyl)oxime (Table 1.1, entry 20) and polymerized it using similar conditions as described above. High-molecular-weight glycopolymers were obtained that displayed narrow polydispersities, a feature attributed to the processing method: precipitation into methanol and two-stage thermal degradation [27]. Similar glycomonomers (Table 1.1, entry 21) containing a urea linkage were polymerized by the same researchers, with the resulting polymers displaying multimodal molecular weight distributions and high glass transition temperatures due to the presence of urea [28]. Kobayashi et al. prepared styrene derivatives with maltose, lactose, and maltotriose substituents on each benzene ring (Table 1.1, entries 22–24) by coupling the corresponding oligosaccharide lactones with p-vinylbenzylamine [13b]. The monomers were then polymerized in either dimethyl sulfoxide (DMSO) using AIBN or in water using potassium peroxydisulfate at 60◦ C. The maltose- and maltotriose-containing polymers interacted specifically with Con A. Kobayashi et al. reported the synthesis of other types of p-vinylbenzamide glycoside derivatives (Table 1.1, entries 25–26). They were homo- and copolymerized with acrylamide using 2,2 -azobisisobutyronitrile (AIBN) as initiator in DMSO at 60◦ C [29]. They also investigated the interaction of the glycopolymers with lectins by means of a two-dimensional immunodiffusion test in agar and inhibition of the

CONVENTIONAL FREE RADICAL POLYMERIZATION

19

hemagglutinating activity. It was found that the specificity of lectins with these glycopolymers was similar to that reported for naturally occurring glycoconjugates and binding between wheat germ agglutinin lectin (WGA) and poly[(p-vinylbenzamido)␤-diacetylchitobiose] was increased by 103 times compared with that of the oligosaccharide itself. Similar monomers (Table 1.1, entries 27–29) have also been synthesized by Akaike and co-workers and polymerized with AIBN in DMSO [30]. They investigated the specific binding of the glucose-derivatized polymers to the asialogylcoprotein receptor of mouse primary hepatocytes. More recently, Nishida and co-workers reported the synthesis of novel vinyl monomers (Table 1.1, entries 30–31) bearing galacto-trehalose (GT), a novel class of 1,1 -linked nonreducing disaccharide possessing an ␣-galactoside epitope. The monomers were copolymerized with acrylamide, and the resulting glycopolymers showed specific binding to ␣-galactoside-specific proteins (BSI-B4 lectin and Shiga toxin-1) [31]. 1.3.4 Other Vinyl-Containing Glycomonomers Monomers derived from N-acetyl-d-glucosamine (Table 1.1, entry 32) and N-acetyld-lactosamine (Table 1.1, entry 33) and chitobiose (Table 1.1, entry 34) were synthesized by Nishimura and co-workers [32]. They were polymerized with acrylamide in deionized water in the presence of ammonium peroxodisulfate (APS) and tetramethylethylenediamine (TMEDA) at room temperature. The synthetic glycopolymers exhibited good solubility in water and specific adhesion to rat hepatocytes. This research group later reported the preparation of a trisaccharide monomer with a Lex structure, n-pentenyl-O-(␤-d-galactopyranosyl)-(1-4)-[O-(␣-l-fucopyranosyl)(1-3)]-2-acetamido-2-deoxy-␤-d-glucopyranoside (Table 1.1, entry 35) [33]. The monomer was then polymerized under the same conditions as described earlier. A monomer based on vinyl ketone, 7,8-didesoxy-1,2:3,4-di-O-isopopylidene-␣d-galacto-oct-7-ene-1,5-pyranose-6-ulose (Table 1.1, entry 36) was copolymerized with a range of other monomers to generate hydrophilic surfaces. The reactivity ratios of different glycomonomers with methyl methacrylate (MMA), styrene, and acrylonitrile were determined [34]. Chemoenzymatically synthesized matitol- and lactitol-based glycomonomers (Table 1.1, entries 37–38) were polymerized to afford glucose-containing and galactosecontaining polymers. The glycopolymers showed specific biological activities toward Con A and RCA120 . Furthermore, positive adhesion to hepatocytes was also observed [35]. The solvent and oxygen effects on the free radical polymerization of 6-Ovinyladipoyl-d-glucopyranose (6-O-VAGlc; Table 1.1, entry 39) were recently investigated by Albertin and co-workers [36]. They polymerized 6-O-VAGlc in water and different alcohols at 60◦ C in the presence of 4,4 -azobis(cyanopentanoic acid) and found that in all cases long polymerization times (>24 h) were necessary to achieve reasonable conversions, and oxygen removal was critical for the success of the experiments.

20

SYNTHESIS OF GLYCOPOLYMERS

1.4 CONTROLLED/LIVING RADICAL POLYMERIZATION Controlled/living radical polymerization techniques have received widespread interest in recent years due to their ability to produce polymers with precise architectures, predefined compositions, and narrow molecular weight distributions, all of which are inaccessible via conventional radical polymerization. The term living polymerization implies that irreversible chain transfer and termination events are absent, a condition that is not strictly upheld in controlled/living radical polymerization since termination events are unavoidable; however, many of the features commonly associated with living polymerization are still attained [37]. Living characteristics include: r The linear evolution of molecular weight with monomer conversion r A constant concentration of active species, which is indicated by a linear plot of ln([M]0 /[M]t ) vs. time r Narrow molecular weight distributions, with the polydispersity index (PDI = Mw /Mn ) remaining below 1.2; a conventional radical polymerization in which termination occurs exclusively by combination gives a theoretical minimum PDI of 1.5 r The ability to polymerize until all monomer is consumed and continue chain growth with the addition of more monomer due to the retention of active end groups Glycopolymers have been synthesized in a controlled/living fashion using the stable free radical polymerization techniques nitroxide-mediated polymerization (NMP) and cyanoxyl-mediated polymerization, the atom transfer radical polymerization (ATRP) technique, and the reversible addition–fragmentation chain transfer (RAFT) technique. 1.4.1 Stable Free Radical Polymerization

1.4.1.1 Nitroxide-Mediated Polymerization Nitroxide-mediated polymerization was developed as a controlled polymerization technique by Solomon et al. in 1985 [38], but it was not until the end of the 1990s that its potential for the synthesis of well-defined glycopolymers was finally realized. Nitroxide-mediated polymerization relies on the reversible capping of growing radical chains with nitroxide species, which are known as persistent radicals (2 in Scheme 1.1). The nitroxides themselves kt Pn

R O N R'

kact k deact

Pn

Dead polymer +

R O N R'

M k p 1

2

SCHEME 1.1 Mechanism of NMP.

21

CONTROLLED/LIVING RADICAL POLYMERIZATION

are not capable of initiating polymerization but instead serve to reduce the active radical concentration in the system and thereby limit bimolecular termination events. The initiating radicals can originate from the fragmentation of an alkoxyamine (as in Scheme 1.1) or can be provided by another source such as a conventional free radical initiator; in the latter case, addition of a nitroxide rather than the corresponding alkoxyamine is the most suitable strategy for controlling the polymerization. Nitroxides are often commercially available as stable radical species. Additives known as accelerators are also commonly employed in NMP and serve the role of regulating the concentration of free nitroxide in the system, which would otherwise build up and retard the polymerization as propagating radicals are inevitably lost through termination. Sulfonic acids, conventional radical initiators, and unstable nitroxides are often used for this purpose [39]. Controlling agents used for the NMP of glycomonomers are shown in Figure 1.1. N1 (TEMPO, a very common controlling agent), N2, N6, and N10 are nitroxides, whereas the remaining species are alkoxyamines, which fragment to generate their corresponding nitroxides. Detailed discussion on the affect of nitroxide structure on NMP kinetics and monomer choice is provided elsewhere [40]. The range of monomers that can be polymerized using NMP is more restricted than other controlled polymerization techniques, and glycopolymer syntheses have largely involved styrenic and acrylic monomers. The majority are protected monomers. Table 1.2 lists the glycomonomers polymerized by NMP. Ohno et al. were the first researchers to synthesize well-defined glycopolymers using NMP. N-(p-vinylbenzyl)-[O-␤-d-galactopyranosyl-(1-4)]-d-gluconamide (VLA; Table 1.2, entry 1) and its acetylated equivalent (AcVLA; Table 1.2, entry 2) were polymerized in N,N-dimethylformamide (DMF) at 90–105◦ C using N3 and a radical initiator as accelerator [41]. Initial attempts to polymerize VLA using N1 were unsuccessful due to partial decomposition of the monomer at the temperatures required for

N1

O

N3

N2

N

O

2,2,6,6-tetramethyl-1-piperidynyl-N-oxy (TEMPO)

O

N

O

O

N

O

O

N

N7

O

N

N

O

di-tert-butyl nitroxide (DBN) O

N6

N5

N4

N

N9

N8

N O

C18H37 N C18H37

C18H37 N C18H37

O

N O O

N

O P O OH O

N10

O

N O P O O

FIGURE 1.1 Nitroxide and alkoxyamine species used for NMP of glycomonomers.

22 1

2

3

Lactose

Glucose

OH

O O HO

OH

HO O

OH NH

O OAc AcO

O

OAc

O

OAc AcO

NH

O O O

O

3-O-acryloyl-1,2:5,6-di-O-isopropylidine-D-glucofuranose (AIpGlc)

O O

O

N-(p-vinylbenzyl)-2,3,5,6-tetra-O-acetyl-4-O-(2,3,4,6-tetraO-acetyl- -D-galactopyranosyl)-D-gluconamide (AcVLA)

AcO

AcOOAc

(1-4)]-D-gluconamide (VLA)

N-(p-vinylbenzyl)-[O- -D-galactopyranosyl-

HO

HOOH

Entry Monomer

Lactose

Sugar

TABLE 1.2 Glycomonomers Polymerized Using NMP

DMF p-xylene

DMF 1,2-dichloroethane

DMF

Solventa

105◦ C 100◦ C

105◦ C 90◦ C

105◦ C

N5, N6 N3

N3 N4

N3

Temperature Initiator

— DCP

DCP DCP

DCP

43 44

41 42

41

Acceleratorb Reference

23

4

5

Gluconolactone

Mannitol

O O O

CH OH

2,3-isopropylidine-1-(4-vinylphenyl) -D-threo(D-erythro)triol

CH,OH O O

2,3;4,5-diisopropylidine-1(4-vinylphenyl)-Dgluco(D-manno)pentitol

O

130◦ C

130◦ C

Bulk, diphenyl ether

Bulk

120◦ C

Bulk

N7

DCP

Poly(styrene)- — N1 N7 DCP

(continued)

46

46

45

24 6

7

8

Fructose

Galactose

O O

CH OH O O O

CH OH

OAc

OAc

O O

4-vinylbenzyl glucoside peracetate

AcO AcO

2,3;4,5-di-O-isopropylidine-1(4-vinylphenyl)-D-manno(D-gluco)hexulo-2,6-pyranose

O O

O O

1,2;3,4-di-O-isopropylidine-1-(4-vinylphenyl)D-glycero(L-glycero)- -D-galactopyranose

Entry Monomer

Galactose

Sugar

TABLE 1.2 Glycomonomers Polymerized Using NMP (Continued)

Chlorobenzene m-xylene

Bulk

Bulk

Solventa

125◦ C 138◦ C

130◦ C

130◦ C

DCP

DCP

47a 47b

46

46

Acceleratorb Reference

N8 CSA Poly(styrene)- — N1

N7

N7

Temperature Initiator

25

b DCP

AcO

O O AcO

O

4

AcO

OAc

O AcO

OAc

AcO

O O O

O

OAc

O

2-(2′,3′,4′,6′-tetra-O-acetyl- -D-galactosyloxy) ethyl methacryl (AcGalEMA)

AcO

AcOOAc

4-vinylbenzyl maltohexaoside peracetate

AcO AcO

OAc

= N,N-dimethyl formamide. = dicumyl peroxide; CSA = (1S)-(+)-10-camphorsulfonic acid.

10

Galactose

a DMF

9

Maltohexaose

O

Dioxane

m-xylene

85◦ C

138◦ C

N9, N10

—

Poly(styrene)- — N1

48

47b

26

SYNTHESIS OF GLYCOPOLYMERS

sufficiently rapid dissociation of TEMPO (> 120◦ C). In contrast, N3 proved effective at lower temperatures (90–105◦ C) due to its higher dissociation constant. Polymerization of VLA using various concentrations of N3 was reasonably well controlled at high N3 concentrations (targeting lower molecular weights); however, high conversions could not be reached, PDIs increased with conversion, and no polymerization occurred when targeting higher molecular weights (target DPn > 100). The addition of the radical initiator dicumyl peroxide (DCP) accelerated the polymerization rate (as expected) without significantly increasing the number of polymer chains in the system; PDIs were unaffected, but the same limiting conversions were observed. Substitution of VLA with its protected analog AcVLA transformed the system into a highly living one, with PDIs consistently around 1.10 and 90% conversion attainable in short polymerization time. Again, the radical initiator greatly enhanced the polymerization rate without affecting the degree of control. Interestingly, the undesirable features of the VLA polymerization are attributed to side reactions, in particular chain transfer to the unprotected hydroxyl groups of the monomer; however, numerous unprotected glycomonomers have since been successfully polymerized using other controlled radical polymerization techniques without such reactions occurring. Despite this discrepancy, almost all subsequent glycopolymer syntheses by NMP have been restricted to protected monomers. The same authors polymerized AcVLA using N4, an alkoxyamine with two long alkyl chains, to generate a lipophilic glycopolymer (Fig. 1.2) [42]. The polymerization was well controlled in 1,2-dichloroethane at 90◦ C, and after deprotection of the acetyl groups using hydrazine the resulting polymer formed a stable liposome when mixed with phospholipid. The liposomes specifically bound the galactose-specific lectin RCA120 . A similar approach was adopted by G¨otz et al., who polymerized 3-O-acryloyl-1,2:5,6-di-O-isopropylidine-d-glucofuranose (AIpGlc; Table 1.2, entry 3) in DMF at 105◦ C using N5 [43]. PDIs remained between 1.06 and 1.20, and the molecular weight increased linearly with conversion. Copolymerization of the glycomonomer with a lipophilic acrylamide monomer was also an effective strategy to generate amphiphilic lipo-glycopolymers. Polymerizations of AIpGlc in p-xylene at 100◦ C using both N3- and an N2-capped poly(styrene) macroinitiator were deemed “living” by Ohno et al., although the level

O

N

AcVLA 1,2-Dichloroethane Dicumyl peroxide 90°C

H C CH

O N

CH

Deprotection

H C CH

H C

CH

N O C H

C H

N O C H

HN OAc

N O C H

O OAc

AcO

O OH

O OH

O OAc O OAc AcO

HN OH

HO

O AcO AcO

O N n

n

C H C H

CH

OH

HO HO HO

FIGURE 1.2 Lipophilic glycopolymer synthesis by Ohno et al. [42].

27

CONTROLLED/LIVING RADICAL POLYMERIZATION

of control more closely resembled that of the unprotected monomer VLA than the protected AcVLA; even in the best case the PDI climbed beyond 1.6 at only 25% conversion, and high molecular weights were unattainable [44]. Despite the questionable “livingness” of the process, deprotection of the isopropylidine groups using formic acid generated amphiphilic diblock copolymers that exhibited microphase separation when cast as thin films. Chen and Wulff produced diblock copolymers of styrene and 2,3;4,5-di-Oisopropylidine-1-(4-vinylphenyl)-d-gluco(d-manno)pentitol (Table 1.2, entry 4) and showed that either block could be polymerized first [45]. N7 (a TEMPO-derived alkoxyamine) was chosen as the mediating agent, and in contrast to the observations of Ohno et al. using VLA, no thermal decomposition of this monomer was noted even after polymerizing for 48 h at 130◦ C. The glycopolymer PDIs were around 1.33, and increased to 1.35–1.80 with the addition of different length poly(styrene) segments. After deprotection, films cast from the block copolymers showed surface properties that varied with the block lengths of the two components. A more extensive kinetic investigation into the N7-mediated polymerization of this and three other styrene-based glycomonomers 2,3-isopropylidine-1-(4-vinylphenyl)-d-threo(d-erythro)triol (Table 1.2, entry 5), 1,2;3,4-di-O-isopropylidine-1-(4-vinylphenyl)-d-glycero(l-glycero)␣-d-galactopyranose (Table 1.2, entry 6), and 2,3;4,5-di-O-isopropylidine-1-(4vinylphenyl)-d-manno(d-gluco)-hexulo-2,6-pyranose (Table 1.2, entry 7), was performed by the same authors [46]. Polymerizations were conducted at 130◦ C in bulk without any monomer degradation; thermogravimetric analysis confirmed that the monomers are thermally stable up to 150◦ C. The polymerization of all four monomers saw a linear increase in Mn with conversion, and PDIs were low (1.24–1.28) when targeting low molecular weights, except in the case of entry 7 whose PDI was inexplicably large (> 1.50). High conversions were attainable for all but monomer 6, which was attributed to higher steric hindrance around its vinyl group. It is interesting to note that the presence of a free hydroxyl group in each monomer does not appear to have compromised the degree of control. Narumi et al. extended the NMP process to synthesize triblock copolymers of 4-vinylbenzyl glucoside peracetate (Table 1.2, entry 8) and styrene using N8, a difunctional TEMPO-based mediator [47a]. Polymerization of the glycomonomer was performed in chlorobenzene at 125◦ C for 5 h using (1S)-(+)-10-camphorsulfonic acid (CSA) as an accelerator to give a well-defined polymer (Mn 8.5 kDa, PDI 1.09) whose two terminal TEMPO groups were used to chain extend with styrene (Fig. 1.3). Small low-molecular-weight shoulders were observed in the size exclusion chromatography (SEC) traces when the polymerization time extended beyond 3 h, but

N O

Ch orobenzene 10-Camphorsulfonic acid 125°C

OAc AcO AcO

O O OAc

N O

CH CH

CH

CH

OAc AcO AcO

O O OAc

CH CH

O N m/2

Styrene Ch orobenzene 125°C

CH

CH

CH m/2

OAc AcO AcO

O O OAc

FIGURE 1.3 Triblock copolymer synthesis by Narumi et al. [47].

CH

O N n/2

28

SYNTHESIS OF GLYCOPOLYMERS

restricting the polymerization time allowed generation of well-defined triblocks (Mn 26 kDa, PDI 1.17). The same monomer was used in the synthesis of star poly(styrene) with a glycopolymer core [47b]. Chains of well-defined poly(styrene) with TEMPO end groups were linked together by chain extending using divinylbenzene with either 4-vinylbenzyl glucoside peracetate or 4-vinylbenzyl maltohexaoside peracetate (Table 1.2, entry 9), thereby incorporating sugar groups into the core. The final star polymers had on average 18 arms, PDIs around 1.35, and after alkaline deprotection of the sugar groups were capable of encapsulating water-soluble molecules in chloroform solution. Ting et al. published the most recent report on NMP of a sugar monomer, namely 2(2 ,3 ,4 ,6 -tetra-O-acetyl-␤-d-galactosyloxy)ethyl methacrylate (AcGalEMA; Table 1.2, entry 10), which is the first report detailing the polymerization of a methacrylic sugar monomer by NMP [48]. Difficulties in polymerizing methacrylates by NMP (such as enhanced disproportionation between growing radicals and nitroxide) were overcome by copolymerizing with a small proportion of styrene, which reduced the average activation–deactivation constant and thereby suppressed the irreversible termination events. The commercially available alkoxyamine N9 (which contains an N10 nitroxide end group) was used to mediate the polymerization at 85◦ C in dioxane to generate the random copolymer poly(AcGalEMA-co-styrene), which exhibited linear evolution of molecular weight with conversion and a final PDI of 1.26. However, chain extension of this block with styrene at 115◦ C resulted in some low-molecular-weight tailing, which was attributed to loss of alkoxyamine end groups. Alternatively, chain extension of a well-defined poly(styrene) homopolymer with glycomonomer/styrene furnished the block copolymer with a PDI of 1.15. The polymerization was commenced at 120◦ C to encourage cleavage of the poly(styrene)-N10 macroinitiator before reducing the temperature to 85◦ C for the remainder of the polymerization, since the activation–deactivation constant for the methacrylate-based alkoxyamine is higher than that for styrene. Deprotection of the galactose acetyl groups afforded amphiphilic block copolymers capable of forming bioactive micelles and porous films.

1.4.1.2 Cyanoxyl-Mediated Polymerization Cyanoxyl radicals, NCO• , were found by Druliner to act as persistent radicals capable of mediating the polymerization of acrylates, methacrylates, and methacrylamides [49]. The NCO• radicals are generated in situ by the one-electron reduction of cyanate anions using arenediazonium ions (e.g., p-chlorobenzenediazonium cations, Scheme 1.2). The aryl radicals produced simultaneously initiate polymerization, and their associated cyanoxyl radicals (which are not capable of initiating polymerization) act as reversible capping agents to form dormant species that can be reactivated by homolytic cleavage of the C–O bond. The C–O bonds in these systems are more easily cleaved than the equivalent bonds in alkoxyamines, which offers a distinct advantage over NMP in the polymerization of thermally sensitive glycomonomers; cyanoxyl-mediated polymerizations are typically conducted from 25 to 70◦ C. In addition, tolerance to a wide range of functional groups including hydroxyls, amines, and carboxylic acids broadens the scope of potential monomer–solvent combinations. It must be noted, however,

29

CONTROLLED/LIVING RADICAL POLYMERIZATION

N N+

Cl

+

-

OC

N

N2

+

Cl

OC

N

Dead polymer

X kt kact Cl

CH2

CH CH2

CH OC N

X

X

n

kdeact

Cl

CH2

CH CH2

CH

X

X

n

+

OC

N

X kp

SCHEME 1.2 Mechanism of cyanoxyl-mediated polymerization.

that while cyanoxyl radicals introduce some degree of mediation, the inability to target predefined molecular weights by stopping the polymerization at a particular time precludes cyanoxyl-mediated polymerization systems from being considered truly living/controlled. Low initiator efficiency due to irreversible termination of aryl radicals in the initial stages of the polymerization is responsible for this lack of predictability in molecular weights [50]. Chaikof’s work group pioneered the application of cyanoxyl-mediated polymerization for the synthesis of glycopolymers from unprotected monomers. Cyanoxyl radicals were generated in situ by the reduction of cyanate using pchlorobenzenediazonium cations, simultaneously producing p-chlorobenzene radicals that initiated polymerization (Scheme 1.2). The glycoolymers were designed as heparin and heparin sulfate mimetics, with later publications focusing on their various biomimetic capabilities. In their early work, nonsulfated and sulfated N-acetyl-d-glucosamine-based monomers with two different spacer arms between the vinyl group and the sugar (Table 1.3, entries 1–2) were copolymerized with acrylamide to generate statistical copolymers with different proportions of sugar groups [51]. The molecular weights increased with conversion and the final statistical copolymers displayed PDIs ranging from 1.10 to 1.47. A higher glycomonomer feed ratio did compromise the level of control, presumably due to the lower reactivity of the unactivated vinyl group. Indeed, further investigation revealed that homopolymerization of these alkene-derivatized monomers was not possible, prompting the development of acrylate alternatives (Table 1.3, entries 3–4) that were capable of homopolymerization [50]. It is interesting to note, however, that copolymers of these nonsulfated and sulfated acrylates with acrylamide generally displayed higher PDIs than those synthesized using the alkene glycomonomers, potentially due to the higher frequency of termination events in acrylate systems. Extension of the same synthetic protocol to include alkene-derivatized nonsulfated and sulfated lactose monomers (Table 1.3, entries 5–6) broadened the library of copolymers to test for heparin-like abilities [52]. In vivo, heparin sulfate is known to bind to fibroblast growth factor 2 (FGF-2) and promote its binding to FGF receptor1 (FGFR-1). When tested in this capacity, polymers containing either sulfated

30 1

2

3

Glucosamine

Glucosamine

O NHAc

O n = 3, 9

O NHAc

O n = 3, 9

NHAc

O O O

O

(4,5-Dihydroxy-6-hydroxymethyl3-methyl-carboxamidotetrahydro-2H-2pyranyloxy)ethyl acrylate

HO HO

OH

n=3: n-Pentenyl 2-acetamido-2-deoxy3,4,6-trisulfoxy-D-glucopyranoside n=9: n-Undecenyl 2-acetamido-2-deoxy3,4,6-trisulfoxy-D-glucopyranoside

O3SO – O3SO

–

OSO3–

n=3: n-Pentenyl 2-acetamido-2deoxy-D-glucopyranoside n=9: n-Undecenyl 2-acetamido-2deoxy-D-gluco pyranoside

HO HO

OH

Entry Monomer

Glucosamine

Sugar

H2 O/THF

H2 O

H2 O

Solventa

TABLE 1.3 Glycomonomers Polymerized by Cyanoxyl-Mediated Polymerization

50◦ C

50◦ C

50◦ C

ClC6 H4 N≡N+ BF4 − /NaOCN

ClC6 H4 N≡N+ BF4 − /NaOCN

ClC6 H4 N≡N+ BF4 − /NaOCN

Temperature Initiating System

50, 54

50–52

50–52

Reference

31

4

5

6

Glucosamine

Lactose

Lactose

O3SO – O3SO O NHAc

O O

O

OH

O O HO

OH

HO

O O

O3SO –

–

O O3SO

O3SOOSO3 –

O O3SO –

O O3SO

OSO3– O

n-Pentenyl O-(2,3,4,6-tetra-O-sulfo- -D-galactopyranosyl)(1-4)-2,3,6-tri-O-sulfo- -D-glucopyranoside

–

–

n-Pentenyl O-( -D-galactopyranosyl)-(1-4)-D-glucopyranoside

HO

HOOH

(4,5-Disulfoxy-6-sulfoxymethyl-3-methylcarboxamidotetrahydro-2H-2-pyranyloxy) ethyl acrylate

–

OSO3–

H2 O/THF

H2 O/THF

H2 O/THF, H2 O

50◦ C

50◦ C

50◦ C

ClC6 H4 N≡N+ BF4 − /NaOCN

ClC6 H4 N≡N+ BF4 − /NaOCN

(continued)

52

52

ClC6 H4 N≡N+ BF4 − /NaOCN 50, 53, 54

32

= tetrahydrofuran.

8

Lactose

a THF

7

OH

O O HO

OH

HO

O O N H

O

O3SO

–

O O O3SO

– –

O O3SO

OSO3– O N H

O

2-N-Acryloyl-aminoethoxyl-4-O-(2,3,4,6-tetra-O-sulfo- -Dgalactopyranosyl)-(1-4)-2,3,6-tri-O-sulfo- -D-glucopyranoside

O3SO

–

–

O3SO OSO3

–

2-N-Acryloyl-aminoethoxyl-4-O-( -D-galactopyranosyl)-(1-4)-D-glucopyranoside

HO

HOOH

Entry Monomer

Lactose

Sugar

H2 O

H2 O

H2 O 1:1 H2 O/THF H2 O

Solventa

65◦ C

50◦ C

50◦ C 50◦ C 65◦ C

BiotinC6 H4 N≡N+ BF4 /NaOCN VariousC6 H4 N≡N+ BF4 /NaOCN

ClC6 H4 N≡N+ BF4 − /NaOCN BiotinC6 H4 N≡N+ BF4 /NaOCN VariousC6 H4 N≡N+ BF4 /NaOCN

Temperature Initiating System

TABLE 1.3 Glycomonomers Polymerized by Cyanoxyl-Mediated Polymerization (Continued)

56

53, 54

54 55 56

Reference

33

CONTROLLED/LIVING RADICAL POLYMERIZATION

RO OR

OR O

RO OR

O

O

O RO

O RO + O

N H

Biotin Biotin C H N N BF /NaOCN 1:1 H O/THF, 50°C

CH

CH O

RO OR

co CH

CH O

NH

OR O

RO

H N

NH

OR

O RO

OC N n

O O RO

R = H / SO

FIGURE 1.4 Synthesis of biotinylated statistical copolymers by Hou et al. [56].

N-acetyl-d-glucosamine or sulfated lactose groups showed a pronounced enhancement in the binding of FGF-2 to FGFR-1, particularly for the lactose candidate, compared to their nonsulfated counterparts. Interestingly, the linker length had no influence. Further investigation into the chaperone ability of sulfated lactose/acrylamide copolymers found that a particular copolymer composition (Mn 9.3 kDa, 10 mol% glycomonomer) was as effective as heparin sulfate in protecting FGF-2 from enzymatic, acidic, and thermal degradation and for promoting binding to FGFR-1 [53]. Acrylamide-based lactose monomers (Table 1.3, entries 7–8) were copolymerized with acrylamide in an identical fashion to above, giving statistical copolymers with PDIs ranging from 1.19 to 1.47 [54]. Polymers containing sulfated lactose groups demonstrated a significant anticoagulation ability, whereas nonsulfated lactose polymers and neither sulfated nor nonsulfated N-acetyl-d-glucosamine-containing polymers showed any effect. Interestingly, sulfated lactose homopolymers were outperformed by their copolymers with acrylamide. End-functionalization of glycopolymers synthesised by cyanoxyl-mediated polymerization was also reported by Chaikof’s work group. Two biotin-derivatized arylamine initiators (each with different spacer lengths between the biotin and aryl groups) were used to generate biotin-terminated lactose/acrylamide copolymers that interacted strongly with streptavidin (irrespective of spacer length) [55]. Elaborating on this concept, a series of arylamine initiators derivatized with alkoxy, amino and Fmoc-protected amino, biotinyl, hydrazido, and carboxyl functionalities were effective in mediating the familiar glycomonomer/acrylamide copolymerizations (Fig. 1.4) [56]. The unprotected amino species and the hydrazine-functionalized species both gave lower yields, a feature that was attributed to quaternization of the amino group that suppressed radical formation. The series of end-functionalized glycopolymers were promising candidates for various bioconjugation reactions. 1.4.2 Atom Transfer Radical Polymerization Atom transfer radical polymerization (ATRP, Scheme 1.3) is a controlled radical polymerization technique that was developed independently by the research groups headed by Sawamoto [57] and Matyjaszewski [58] in 1995. An ATRP system contains a halogenated organic compound (initiator) R–X, a transition metal Mn , which can increase its oxidation number, a complexing ligand L to stabilize the metal, and monomer M. Initiation involves abstraction of the initiator’s halogen atom by the metal complex, which simultaneously undergoes a single electron oxidation.

34

SYNTHESIS OF GLYCOPOLYMERS

R X

+

Mn / L

k act k de act

Mn+1X / L

+

R

kt

Dead polymer

M k p

SCHEME 1.3 ATRP mechanism.

This gives an organic radical R• and a new metal complex Mn+1 X/L in which the metal’s oxidation number has increased by 1. The radical can add monomer units, thereby initiating chain growth, before abstracting the hydrogen atom from the metal complex and restoring its dormant state Pn –X. This halogen-capped polymer chain assumes the same role that the initiator occupies in the initiation step; it remains in its dormant state until activated by the metal complex to reform the radical Pn • , which can then add a few more monomer units before being deactivated. These atom transfer equilibria lie heavily toward the dormant species, which reduces the effective radical concentration and thereby limits bimolecular termination. Termination still occurs but is greatly suppressed, imparting living characteristics on the polymerization process. Atom transfer radical polymerization has been successfully performed using a variety of transition metals, but copper (Cu) complexes have proven to be the most efficient and versatile. The choice of ligand influences the relative rates of activation and deactivation and, therefore, the degree of control over the polymerization. Nitrogen-containing multidentate ligands are commonly employed for Cu-mediated ATRP, and those relevant to glycopolymer synthesis are shown in Figure 1.5. Figure 1.6 shows the initiators relevant to the present review. For simplicity, the bold code will be used in text to refer to each ligand and initiator, rather than including its full or abbreviated name. In cases where the initiating group was attached to another structure, such as a premade polymer or a surface, the structure will be stated followed by the code of the initiating group. For example, a poly(ε-caprolactone) (PCL) species end-functionalized with an ethyl 2-bromoisobutyryl group A1 will be denoted PCL-A1. The glycomonomers polymerized using ATRP are summarized in order of appearance in Table 1.4, including their polymerization conditions.

1.4.2.1 Linear Polymers Ohno et al. from Kyoto University reported the first ATRP of a sugar monomer by polymerizing 3-O-methacryloyl-1,2:5,6-di-Oisopropylidine-d-glucofuranose (MAIpGlc; Table 1.4, entry 1) using a CuBr/L3 catalyst in veritrole containing A1 as initiator at 80◦ C [59]. The first-order kinetic plots for the polymerizations of MAIpGlc were approximately linear, which means that the system obeys first-order kinetics with respect to monomer concentration. However, a twofold increase in the initiator concentration (holding all else constant) had a negligible effect on the rate of polymerization rp , whereas a doubling of the activator concentration increased rp by a factor of 3. Previous studies using styrene indicated that rp is first order with respect to both the dormant alkyl halide concentration and

CONTROLLED/LIVING RADICAL POLYMERIZATION

L1

N

L2

N

N

L3

N

N

4,4'-Dimethyl-2,2'-bipyridine (DMDP)

2,2'-Bipyridine (bipy)

C7H15

35

L4

C7H15

C10H21

N

C10H21

N

N

4,4'-di-n-Heptyl-2,2'-bipyridine (dHbipy)

4,4'-bis(1-Decyl)-2,2'-bipyridine (dDbipy)

L5

L6 N

N

N

N

N N

N

1,1,4,7,7-Pentamethyldiethylene triamine (PMDETA)

1,1,4,7,10,10-Hexamethyltriethylene tetramine (HMTETA)

L7

L8

N

N N

N

N C8H17

N N-n-octyl-2-pyridylmethanimine

tris(2-(Dimethylamino)ethyl)amine (Me6-TREN)

FIGURE 1.5 Ligands employed Cu(I) catalyst systems for glycopolymer synthesis using ATRP.

A1

A2

A3

O Br

O

Br

Br

Ethyl 2-bromoisobutyrate

A4

Br Dibromoxylene

1-Phenylethyl bromide

A5

O O

Br

Ethyl 2-bromoisopropionate

(MeO)3Si CH2 CH2

O S Cl O

2-(4-Chlorosulfonylphenyl)ethyltrimethoxysilane

FIGURE 1.6 Initiators employed for glycopolymer synthesis using ATRP.

36

1

2

Glucose

O O

O

O

OAc

O O O

O

glucopyranosyloxy)ethyl acrylate (AcGEA)

2-(2′,3′,4′,6′-tetra-O-acetyl- -D-

AcO AcO

OAc

3-O-methacryloyl-1,2:5,6-di-Oisopropylidine-D-glucofuranose (MAIpGlc)

O O

O

Entry Structure

Glucose

Sugar

TABLE 1.4 Glycomonomers Polymerized Using ATRP

Chlorobenzene Chlorobenzene

Anisole Ethyl acetate Veratrole Anisole, ethyl acetate Toluene

Ethyl acetate

Ethyl acetate

Veratrole Ethyl acetate

Solventa

80◦ C

80◦ C

70◦ C

90◦ C 100◦ C 80◦ C 60◦ C

60◦ C, 60◦ C, 100◦ C 60◦ C

60◦ C

80◦ C

A2 A2

N-succinimidyl-A4

25-arm star silsesquioxane-A1 A1-poly(sulfone)-A1 Silica-A1 Silica-A5 Carbon nanotube-A1

A1 poly(2-(A1)ethyl methacrylate) A1

Temperature Initiator

CuBr/L1 CuBr/L5

CuBr/L8

CuBr/L2 Ni(PPh3 )2 Br2 CuBr/L3 CuBr/L6

CuBr/L6, CuBr/L5 Ni(PPh3 )2 Br2 CuBr/L6

CuBr/L3 CuBr/L6

Catalyst

61–63 64

92

82 85 86 87

81

80

59 79

Reference

37

3

4

Glucose

Lactose

OH

O O HO

OH OH H N HO O O

2-Lactobionamidoethyl methacrylate (LAMA)

HO HO

OH

O

O

methacrylate (GAMA)

OH H N HO O

D-gluconamidoethyl

HO HO

OH

O

25◦ C 35◦ C 30◦ C

NMP DMSO

MeOH/H2 O

NMP, 3:2 MeOH/H2 O NMP H2 O, MeOH/H2 O NMP NMP/H2 O

4-Arm PCL-A1 Gold-A1 Biotin-PEG-A1 A1-disulfide-A1

Aldehyde-A1 25◦ C 20◦ C 20◦ C 25◦ C

Titanium-A1 20◦ C

Poly(ethylene oxide)-A1 Poly(ethylene oxide)-A1 4-Arm star PCL-A1 4-Arm poly(peptide)-A1 12-Arm star PCL-A1 A4poly(pseudorotaxane)A4 Membranepoly(HEMA)-A4 Gold-A1 25◦ C

20◦ C

25◦ C 25◦ C

NMP NMP

H2 O, MeOH/H2 O 3:2 MeOH/H2 O

20◦ C

20◦ C

MeOH, MeOH/ H2 O, H2 O MeOH

CuBr/L1 CuBr/L1 CuBr/L1 CuBr/L1

CuBr/L1

CuBr/CuBr2 /L1

CuBr/L1

CuBr/CuBr2 /L1

CuBr/L1 CuBr/L5

CuBr/L1 CuBr/L1

CuBr/L1

CuBr/L1

(continued)

75 89 95 98b

65b, 66

90

89

88

77 78

74 76

66

65a

38

5

6

7

8

Lactose

Glucose

Glucose

O

O O

O OAc AcO

O

OAc

AcO

O O O

O

O O

O

O

OH

O O O

O

2-methacryloxyethyl glucoside

HO HO

OH

3-O-acryloyl-1,2:5,6-di-O-isopropylidine-Dglucofuranose (AIpGlc)

O O

O

2-O-acryloyloxyethyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)(1-4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (AEL)

AcO

AcOOAc

6-O-methacryloyl-1,2;3,4-di-Oisopropylidine-D-galactopyranose (MAIpGal)

O

OO

O

Entry Structure

Galactose

Sugar

TABLE 1.4 Glycomonomers Polymerized Using ATRP (Continued)

3:2 MeOH/H2 O

Ethyl acetate

Chlorobenzene

THF Toluene

Anisole Chlorobenzene

Solventa

25◦ C

60◦ C

100◦ C

90◦ C 60◦ C, 60◦ C, 25◦ C 60◦ C 70◦ C

Azide-A1

A1

A3

A1-PCL-A1 A4, A1, A1 PCL-A1 N-succinimidyl-A4

Temperature Initiator

CuBr/L1

CuBr/L5

CuBr/L1

CuBr/L4 CuCl/L1, CuBr/L1, CuBr/L5 CuBr/L6 CuBr/L8

Catalyst

91

83, 84

73

72 92

70 71

Reference

39

11

12

Glucosamine

Mannose

O

O O

OH HO O O

O NHAc

O

O NHAc

O

O

O

O

O

O

O

(MEMan)

2-methacryloxyethyl-D-mannopyranoside

HO HO

HO HO

OH

AcO AcO

OAc

6-O-(4-vinylbenzyl)-1,2:3,4-di-O-isopropylidineD-galactopyranose (VBIG)

O

OO

MeOH

85:15 MeOH/H2 O 3:1 MeOH/H2 O

DMSO, MeOH

DMSO, MeOH

Chlorobenzene

MeOH = methanol; NMP = N-methyl-2-pyrrolidone; DMSO = dimethyl sulfoxide; THF = tetrahydrofuran.

10

Glucosamine

a

9

Galactose

Pyrene-PCL-A1

Peptide-A1 Pyridyl disulfide-A1 A1-disulfide-A1

23◦ C 30◦ C 25◦ C

25◦ C, 30◦ C Biotin-A1

25◦ C, 30◦ C Biotin-A1

115◦ C

CuBr/L1

CuBr/CuBr2 /L1

CuBr/L7, CuBr/L1 CuBr/L1

CuBr/L7, CuBr/L1

CuBr/L1

98a

97

96

94

94

93

40

SYNTHESIS OF GLYCOPOLYMERS

the activator concentration [60]. Despite these unexpected kinetic features, the polydispersity index (PDI) of the resulting glycopolymers was less than 1.30, indicating that the polymerizations were reasonably well controlled. Liang and co-workers were one of the first research groups to generate well-defined glycopolymers using ATRP [61]. The polymerization of 2-(2 ,3 ,4 ,6 -tetra-O-acetyl␤-d-glucopyranosyloxy) ethyl acrylate (AcGEA; Table 1.4, entry 2) was performed in chlorobenzene at 80◦ C using CuBr/L1 catalyst and A2 initiator. The polymerization obeyed first-order kinetics up to 70% conversion, after which the rising viscosity caused deviation from linearity. The measured molecular weights agreed with theoretical values and increased linearly with conversion, and the PDIs remained less than 1.4 throughout. Chain extension of a poly(styrene)-A2 macroinitiator under the same conditions generated well-defined block copolymers (PDI 1.31–1.37) as shown in Figure 1.7 [62]. Deprotection of the hydroxyl groups on the sugar units using sodium methoxide afforded amphiphilic block copolymers that self-assembled in water to form spheres, rods, vesicles, tubules, and finally large compound vesicles as the polymer concentration increased from 0.1 to 2.0 wt% [63]. The CuBr/L5-catalyzed ATRP of the same monomer using a poly(ethylene glycol)-A1 (PEG-A1) macroinitiator furnished a well-defined hydrophilic-hydrophilic block copolymer (after deprotection) that assembled into aggregates capable of binding concanavalin A (Con A) [64]. Narain and Armes synthesized the two unprotected monomers 2-gluconamidoethyl methacrylate (GAMA; Table 1.4, entry 3) and 2-lactobionamidoethyl methacrylate (LAMA; Table 1.4, entry 4) by reacting 2-aminoethyl methacrylate with d-gluconolactone [65]. Homopolymerizations were performed at 20◦ C using CuBr/L1 catalyst and a PEO-A1 macroinitiator in different methanol–water solvent combinations. A drastic increase in polymerization rate and a corresponding decline in control was observed when the polymerization medium was changed from pure methanol to 9:1 methanol–water, 3:2 methanol–water, and finally pure water (Fig. 1.8); full conversion was reached in 15 h using methanol (final PDI of 1.19) and only 30 min in water (final PDI of 1.82). [65a] The polymerization of LAMA proceeded with good control in 3:2 methanol–water (final PDI of 1.10) but not in pure water (final PDI of 1.78); its highly polar nature prohibited dissolution in pure methanol. Various GAMA- and LAMA-containing stimuli-responsive block copolymers were synthesized from macroinitiators or by the direct addition of a second methacrylic monomer to the homopolymerization system at complete conversion [66]. These results are consistent with numerous literature reports on the effect of solvent on the ATRP process. Polar solvents generally increase the rate of polymerization CH3

CH

CH2

CH

Br m

AcGEA CuBr/L1 Chlorobenzene 80°C

CH3

CH

CH2

CH

m

CH2

CH

n O

O OAc AcO AcO

O OAc

Br

O

Deprotection

CH3

CH

CH2

CH

m

CH2 OH

HO HO

CH

n O

O O

OH

Br

O

FIGURE 1.7 Synthesis of amphiphilic block copolymers of poly(styrene)-b-poly(2(2 ,3 ,4 ,6 -tetra-O-acetyl-␤-d-glucopyranosyloxy) ethyl acrylate) by Li et al. [63a].

4 Water

3.5

9:1 Methanol/water

2.5

1.8

14000

1.7

12000

1.6

10000

1.5

Mn

Ln([M ]0/[M ])

3

16000

2

8000

1.4

6000

1.3

4000

1.2

0.5

2000

1.1

0

0

1.5 Methanol

1

0

5

10 Time (h)

15

0

20

40

60

80

Mw /Mn

41

CONTROLLED/LIVING RADICAL POLYMERIZATION

1 100

Conversion (%)

FIGURE 1.8 Pseudo-first-order kinetic plots (left) for the ATRP of 2-gluconamidoethyl methacrylate (GAMA) in different solvent systems, and number-average molecular weight and PDI as a function of conversion (right) for the methanol system. In both cases, [GAMA]0 = 1.675 mol L−1 , [GAMA]0 :[PEO-A1]0 :[CuBr] 0 :[L1] 0 = 50:1:1:2 and T = 20◦ C. Reproduced with permission from ref. [66].

by increasing the activation rate coefficient and decreasing the deactivation rate coefficient. Aqueous systems are especially prone to this effect [67]. Matyjaszewski and Xia state that in a bulk or apolar solvent the a copper catalyst complex is neutral [68] but in aqueous systems most likely forms the cationic [Cu(I)(L1)2 ]+ species with halide counterion [69]. The ionic catalyst is far more active than its neutral counterpart and as a result generates a high concentration of active radicals that increases the polymerization rate but also encourages termination events such as radical coupling and disproportionation reactions. The control over the polymerization is therefore compromised.

1.4.2.2 ABA and Star-Block Copolymers Chen and Wulff synthesized triblock copolymers in which the two terminal blocks are the same (denoted ABA triblock copolymers from here onwards) and star block copolymers by polymerizing 6-O-methacryloyl-1,2:3,4-di-O-isopropylidine-d-galactopyranose (MAIpGal; Table 1.4, entry 5) using linear and four-arm poly(ε-caprolactone)-A1 (PCL-A1) macroinitiators [70]. Linear and star PCL species are conveniently synthesized by ring-opening polymerization (ROP) of ε-caprolactone and can be easily functionalized with ATRP initiators. PCL-based macroinitiator species are therefore popular for the synthesis of block copolymers using ATRP. In Chen and Wulff’s work, a complete shift in the molecular weight distribution and PDIs less than 1.19 confirmed successful and controlled chain growth with no evidence of high-molecular-weight products that are sometimes formed in star synthesis by ATRP. A 5:1 Cu(0)/Cu(II) ratio with L4 ligand was used as the catalyst system. A detailed investigation into the effect of initiator, catalyst, ligand, and temperature on the ATRP of MAIpGal was presented by Meng et al. [71]. Due to its higher initiating efficiency, A1 was more effective than A4 in controlling the polymerization using CuBr/L1 catalyst at 60◦ C, although replacing

42

SYNTHESIS OF GLYCOPOLYMERS

CuBr with CuCl did improve the control in the A4-initiated system. A1/CuBr/L5 at room temperature proved equally effective. Suriano et al. demonstrated that copolymerization of MAIpGal with ␣-methoxy, ␻-methacrylate poly(ethylene oxide) using a PCL-A1 macroinitiator is well controlled by CuBr/L6 in tetrahydrofuran (THF) at 60◦ C, generating amphiphilic block copolymers after deprotection [72]. Atom transfer radical polymerization was combined with ring-opening polymerization (ROP) by Dong et al. to generate ABA triblock copolymers of polypeptide-b-poly(2-acryloxyethyl-lactoside)-b-polypeptide [73]. ATRP of the acetyl-protected lactose monomer 2-O-acryloyloxyethyl-(2,3,4,6-tetra-O-acetyl-␤d-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-␤-d-glucopyranoside (AEL; Table 1.4, entry 6) was performed using A3 initiator and CuBr/L1 complex in chlorobenzene at 100◦ C (Fig. 1.9). The absolute molecular weights as determined by multiangle laser light scattering were consistent with predicted values and the PDIs remained between 1.19 and 1.35. The bromo end groups of the isolated polymers were converted to amino groups, and the resulting diamines were used to initiate the ROP of l-alanine N-carboxyanhydride or ␤-benzyl-l-glutamate N-carboxyanhydride. Removal of the acetyl groups on the sugar units using hydrazine generated well-defined triblocks with the central glycopolymer sandwiched between two polypeptide blocks. Dai and Dong synthesized 4-arm stars starting with a 4-arm star poly(εcaprolactone) (SPCL) produced by ROP [74]. The hydroxyl end groups were converted to A4 species and the star was used to polymerize GAMA in N-methyl-2pyrrolidone (NMP) at room temperature employing CuBr/L1 as catalyst. NMP is a dipolar aprotic solvent that is often chosen over water since it effectively dissolves many polar monomers but avoids the poor control that is often observed in aqueous ATRP systems. The PDIs of the final star copolymers were between 1.09 and 1.33 for different block lengths of glycopolymer. Differential scanning calorimetry (DSC) analysis of the block copolymers showed that the crystallinity of the PCL block was greatly reduced by the presence of the glycopolymer, dropping from almost 70% for the SPCL alone to less than 2% after grafting the GAMA. A transition from micelles to wormlike rods to vesicles was noticed in the self-assembly of the star copolymers as the glycopolymer block length decreased. The stars also specifically recognized Con A in solution. A very similar study using a lactose-based monomer was published by the same authors [75], and the protocol was extended to the synthesis of star glycopolymers from a 4-arm polypeptide macroinitiator [76] and a 12-arm PCL macroinitiator [77].

AcAEL CuBr/L1

Br Br

a) End-group conversion

Chlorobenzene 100°C

n/2

O

AcOOAc

O

OAc O

AcO

Br

O OAc AcO

n/2

O

HOOH

O

O N H

H N

H

m/2

O

OH O

O AcO

H N

b) ROP c) Deprotection

HO OH

O HO

O O HO

FIGURE 1.9 Synthesis of the ABA triblock copolymer poly(l-alanine)-b-poly(2acryloxyethyl lactoside)-b-poly(l-alanine) by Dong et al. [73b].

CONTROLLED/LIVING RADICAL POLYMERIZATION

43

A later publication by the same group presented the synthesis of polypseudorotaxane/glycopolymer hybrids [78]. ␣-Cyclodextrin (␣-CD) was threaded onto a difunctional PCL-A4 macroinitiator via a supramolecular inclusion reaction to give a polypseudorotaxane with ATRP-initiating end groups. It is interesting to note that the more efficient and, therefore, preferred ATRP-initiating group A1 was too bulky to penetrate the ␣-CD cavities; therefore, A4 had to be employed. The polypseudorotaxane was used as initiator to polymerize GAMA in DMSO at 35◦ C using CuBr/L5 catalyst. Different [monomer]0 :[initiator]0 ratios were used, giving final polymers with Mn values of 33–39 kDa and PDIs of 1.26–1.56. Wide-angle X-ray diffraction (WAXD) and DSC analysis confirmed that the crystallization of the PCL was completely suppressed by its inclusion into the ␣-CD cavities and that none of the ␣-CD units unthreaded during polymerization. The biohybrids self-assembled in solution to form spherical micelles or vesicles that specifically recognized Con A and not bovine serum albumin (BSA). Molecular “sugar sticks” were synthesized by Muthukrishnan et al. from narrowly dispersed poly(2-hydroxyethyl methacrylate) of 67 and 418 kDa [79]. The hydroxyl groups of these two polymers were converted to A1 groups to give the polyinitiator species poly(2-(2-bromoisobutyryloxy)ethyl methacrylate). In a preliminary study, the effectiveness of three different catalysts in controlling the homopolymerization of MAIpGlc in ethyl acetate using A1 initiator was investigated, finding that (PPh3 )2 NiBr2 at 100◦ C and CuBr/L6 at 60◦ C both demonstrated good control (final polymer PDIs of 1.18 and 1.19, respectively), but CuBr/L5 resulted in broader molecular weight distributions (final polymer PDI of 1.51) [80]. The CuBr/L6 catalyst system was therefore chosen for the sugar stick synthesis. Conversions were limited to 10% in order to obtain well-defined polymer brushes with poly(MAIpGlc) side chains; the high-molecular-weight polyinitiator’s PDI of 1.08 remained unchanged after grafting. However, the cleaved grafted chains displayed PDIs of approximately 1.30, which suggests that initiation was slow and therefore the level of control was not optimal. In addition, the initiator efficiency was lower than expected (0.23 < f < 0.38). Imaging of the brushes using scanning force microscopy (SFM) and cryogenic transmission electron microscopy (cryo-TEM) revealed highly uniform wormlike structures that were not fully stretched due to the relatively low grafting density. Star inorganic–organic hybrids were synthesized in a similar fashion by ATRP of MAIpGlc from an A1-functionalized 25-arm silsesquioxane [81]. ABA triblock copolymers were synthesized by Wang et al. by polymerizing MAIpGlc from an A1-terminated difunctional polysulfone (Mn 5.7 kDa, PDI of 1.34) in anisole at 90◦ C using CuBr/L2 [82]. The final polymer (Mn 12.5 kDa, PDI of 1.18) self-assembled after deprotection of the sugar units to give spherical aggregates in mixed N,N-dimethyl formamide (DMF)–water.

1.4.2.3 Hyperbranched Glycopolymers Hyperbranched glycopolymers were synthesised by Muthukrishnan et al. by using a technique called self-condensing atom transfer radical copolymerization (Fig. 1.10) [83]. The acrylic glycomonomer 3-O-acryloyl-1,2:5,6-di-O-isopropylidine-d-glucofuranose (AIpGlc; Table 1.4, entry 7) was copolymerized with the acrylic AB∗ initiator–monomer (or inimer)

44

SYNTHESIS OF GLYCOPOLYMERS

*M B*

m

*A m

O O O

O

Br

+

O O

b a

O

CuBr/L5 Ethyl Acetate 60°C

O

O

*M

a

m

m

m

M*

m *M

m

m m

a m

B*

a

b

O O

A

m

m

m

b

M

m

a *B

m

b

m

A* B*

m

FIGURE 1.10 Muthukrishnan et al.’s synthesis of hyperbranched structures by copolymerizing the monomer AIpGlc (denoted M in the scheme) with the “initiator–monomer” species 2-(2-bromopropionyloxy)ethyl acrylate (BPEA, denoted A B∗ ). A∗ , B∗ , and M∗ are active groups and a, b, and m are reacted ones [83].

2-(2-bromopropionyloxy)ethyl acrylate (BPEA) using ATRP with the CuBr/L5 catalyst. The copolymerization introduces both AIpGlc units and BPEA units into the polymer chains, and each of the BPEA units is capable of initiating ATRP via its bromo group, which allows the generation of hyperbranched structures without significant crosslinking since the ATRP conditions minimize chain transfer and combination events. Investigation of the homopolymerization of AIpGlc using A1 indicated that a lower reaction temperature of 60◦ C was preferable to avoid the bimodal molecular weight distributions observed at 80 and 100◦ C as a result of recombination events. At 60◦ C in ethyl acetate the homopolymer PDIs remained between 1.09 and 1.14; therefore, 60◦ C was preferred for the generation of the hyperbranched structures. Removal of the isoproylidine groups gave hydrophilic hyperbranched polymers, which were used to generate thin bioactive glycopolymer films on a hydrophobic surface using a low-pressure plasma immobilization technique [84]. Hyperbranched structures based on MAIpGlc were generated in a similar fashion using (PPh3 )2 NiBr2 at 100◦ C and CuBr/L6 at 60◦ C in ethyl acetate [80]; (PPh3 )2 NiBr2 was preferred because it facilitated a higher degree of branching. A silicon wafer functionalized with a bromoester initiator facilitated the grafting of hyperbranched poly(MAIpGlc) from the surface [85]. As expected, the monomer feed ratio ␥ = [monomer]0 /[inimer]0 had a significant effect on the properties and morphologies of the resulting films since the degree of branching declines as less inimer is incorporated. The surface attained an enormous swelling ability once the isopropylidine groups were removed, with the wet surface thickness jumping from 4.6 to 29.9 nm.

1.4.2.4 Surface Grafting Surface-initiated ATRP is an effective method for achieving a high density of polymer brushes covalently linked to a surface, but maintaining a sufficient deactivator concentration during polymerization is crucial in maintaining the balance between the dormant and active species. In a conventional solution or bulk ATRP, sufficient Cu(II) is generated by reaction of the Cu(I) catalyst with the initiator at the start of the polymerization. However, the concentration of initiator in a surface-initiated ATRP is generally insufficient to achieve this purpose,

45

CONTROLLED/LIVING RADICAL POLYMERIZATION

so alternative means are required in order to boost the deactivator concentration. The first approach is to add sacrificial initiator, which means that polymerization proceeds both in solution (initiated by the sacrificial initiator) and from the surface (initiated by the surface-immobilized initiator). The advantage of this approach is the production of free polymer, which is more easily analyzed than the grafted polymer on the surface, and which can often be assumed to represent the characteristics of the grafted polymer. However, the free polymer chains can be adsorbed to the surface of the substrate and interfere with the grafting process. In addition, monomer is consumed not only at the surface but in solution, which may be undesirable if the monomer is precious. The alternative approach is to add Cu(II) to the initial polymerization mixture, which ensures sufficient deactivator is present at the commencement of polymerization. Ejaz et al. used ATRP to functionalize a solid surface with a densely grafted glycopolymer layer (Fig. 1.11) [86]. MAIpGlc was grafted from a silica surface affixed with a monolayer of A5, which was attached using the Langmuir–Blodgett technique. The chlorosulfonylphenyl group of A5 is a highly effective ATRP initiator, and is claimed by the authors to be more capable than A1 at controlling the polymerization of MAIpGlc. In this case, free initiator was added to the system. Despite the bulkiness of the monomer, the thickness of the polymer layer as measured by ellipsometry increased concurrently with the Mn of the free polymer in solution, strongly supporting the proposal that the chains grow from the surface in a controlled fashion. The PDI of the free chains remained less than 1.2 up to high conversion, at which the Mn reached 60 kDa. Quantitative deprotection of the isoproylidine groups gave a surface covered with a high density of well-defined glucose-containing polymer chains. Gao and co-workers grafted linear and hyperbranched MAIpGlc glycopolymers from the surface of multiwalled carbon nanotubes (MWNTs) functionalized with A1 groups using CuBr/L6 at 60◦ C in ethyl acetate [87]. Thermogravimetric analysis (TGA) was used to determine the molecular weight of the polymer on the MWNTs and revealed that Mn of the grafted polymer increased linearly with monomer conversion, although the Mn values were much lower than the expected values due to the low initiating efficiency. Interestingly, the grafted polymer synthesized without sacrificial

Si O Si CH2 CH2

O S Cl O

MAIpGlc CuBr/L3, A5 Veratrole 80°C

Si O Si CH2 CH2

O S O

CH2

CH3 C C O

O O

O

Cl n

O O

Formic Acid Deprotection

Si O Si CH2 CH2

O S O

CH2

O

CH3 C

Cl n

C O OH

O

OH

HO

O OH

FIGURE 1.11 Surface grafting of MAIpGlc from a silica substrate by Ejaz et al. [86].

46

SYNTHESIS OF GLYCOPOLYMERS

initiator demonstrated almost identical kinetics to the system containing the sacrificial initiator. When sacrificial initiator was used, SEC analysis of the free polymer showed the emergence of high-molecular-weight coupling products beyond approximately 45% conversion. This indicates that the MWNTs are not simply an inert scaffold but are actually involved in the polymerization mechanism since a conventional ATRP of the same monomer in an equivalent homogeneous system showed no highmolecular-weight coupling even beyond 80% conversion. This effect was attributed to the “unique electronic property of carbon” but was not explained in further detail. Microscopy of the grafted MWNTs revealed the distinct core–shell structure of the polymer-coated nanotubes. In the same study, hyperbranched glycopolymers were also grafted from the MWNT–A1 using an identical protocol to the group’s previous hyperbranched glycopolymer grafting publications [85]. A polypropylene microporous membrane was grafted with glycopolymer brushes by Yang et al. using a combination of ultraviolet (UV)-induced graft polymerization and ATRP [88]. The UV-promoted polymerization of 2-hydroxyethyl methacrylate (HEMA) introduced a high density of hydroxyl groups to the surface, approximately 62% of which were converted to A4 groups. GAMA was grafted from the substrate using CuBr/L1 catalyst in two different water–methanol mixtures at 30◦ C without the use of sacrificial initiator. A rapid initial polymerization rate followed by a plateau in the number-average degree of polymerization (as determined gravimetrically) was observed in the case where the methanol–water ratio was 3:2. Lowering the water content to 4:1 reduced the polymerization rate and imparted greater control over the polymerization. The addition of CuBr2 , as expected, markedly improved the control. Mateescu and co-workers polymerized GAMA and LAMA from a gold surface functionalized with ␻-mercaptoundecyl bromoisobutyrate (i.e., gold–A1) [89]. Polymerizations were performed at 20◦ C using CuBr/L1 catalyst in water or methanol/water mixtures. Either sacrificial initiator or Cu(II) deactivator were used to provide a sufficient concentration of Cu(II) to control the polymerization, although PDIs of free polymer produced from sacrificial initiator ranged from 1.6–1.8. An increase in the maximum film thickness and a decrease in the polymerization rate were observed as the water content of the polymerization medium decreased, which is consistent with numerous reports on the destabilizing effect of water in the ATRP process. Despite the rather poor control, the poly(GAMA)- and poly(LAMA)-grafted surfaces showed strong binding affinities for Con A and RCA120 , respectively. Raynor et al. performed surface-initiated ATRP of GAMA from a titanium surface functionalized with A1 groups using CuBr/CuBr2 /L1 in methanol–water [90]. The brush thickness increased for the first 4 h but then plateaued due to a loss of ATRP end groups as observed by X-ray photoelectron spectroscopy (XPS). The living behavior of the polymerization was not verified. The glycopolymer-grafted surface resisted protein and cell adhesion.

1.4.2.5 End-Functionalization and Bioconjugation An interesting application of ATRP and click chemistry was reported by Gupta et al. (Fig. 1.12) [91]. An azide-terminated glycopolymer was synthesized by the ATRP of the unprotected monomer 2-methacryloxyethyl glucoside (Table 1.4, entry 8) using an

47

CONTROLLED/LIVING RADICAL POLYMERIZATION OH

O N3

O

O

+

Br

O

O

O

O

HO HO

O

CuBr/L1 3:2 MeOH/H O 25°C

O

OH

N

O

O

Br n

O O

O

OH HO HO O

O

O

N N N

O

excess HN

O O

O

O HN

O OH

Br n

O

O

O

OH

fluorescein

"Click" conditions

O

O

O

HO HO

O OH

COOH

HO

H N

H N

O

O

"Click" conditions

H N

H N

N

N N N

Br n

CPMV

CPMV O

O

150

O

O fluorescein

O R

O 125±12

FIGURE 1.12 Synthesis of virus–glycopolymer conjugates using ATRP and click chemistry by Gupta et al. [91].

azide-terminated A1 derivative with CuBr/L1 in 3:2 methanol–water at 25◦ C to give a relatively well-defined polymer (Mn = 13 kDa and PDI of 1.30). The azideterminated polymer was reacted with an excess of fluorescein dialkyne to give a fluorescently labeled glycopolymer with a single alkyne group at the terminus. Azide groups were then installed on the outer surface of cowpea mosaic virus (CPMV) particles at 150 of the available 240 lysine locations, and 125±12 polymer chains per particle were able to be clicked on via these azide functionalities. This method of attaching polymer chains to virus particles was more efficient than previously reported strategies, and the resulting glycopolymer–CPMV conjugates were investigated further as targeting agents for overexpressed carbohydrate receptors on cancer cells. Indeed, strong and specific binding to both immobilized and free Con A occurred almost instantaneously. An N-(hydroxy)succinimide-terminated A4 initiator was used by Ladmiral et al. to polymerize the protected monomers MAIpGlc and MAIpGal with CuBr/L8 catalyst in toluene at 70◦ C to give well-defined homopolymers [92]. Initiating efficiencies were less than 50% compared to almost 100% for the analogous initiator based on A1, but the less efficient species was preferred because its reactivity toward bioconjugation to peptides and proteins was found to be far superior. Copolymerization of each monomer with a fluorescent methacrylate generated fluorescent statistical copolymers capable of reacting with primary amines courtesy of the terminal N(hydroxy)succinimide groups. A PCL species with a pyrene end group was synthesized by Lu et al., converted to a pyrene–PCL–A1 macroinitiator, and used for the polymerization of 6-O(4-vinylbenzyl)-1,2:3,4-di-O-isopropylidene-d-galactose (VBIG; Table 1.4, entry 9)

48

SYNTHESIS OF GLYCOPOLYMERS

using CuBr/L1 in chlorobenzene at 115◦ C [93]. The block copolymer (Mn 9.0 kDa, PDI 1.28) was deprotected, self-assembled in water, and the glycopolymer shell was crosslinked using a difunctional aldehyde. Hydrolysis of the PCL core under basic conditions was proven by the loss of fluorescence from the pyrene group. V´azquez-Dorbatt and Maynard polymerized both a protected and unprotected monomer (Table 1.4, entries 10–11) synthesized by functionalizing N-acetyl-dglucosamine with 2-hydroxyethyl methacrylate (HEMA). The ATRP was carried out using a biotinylated A1 derivative with CuBr/L7 as catalyst in DMSO at room temperature [94]. The polymerization of the protected monomer was very rapid, reaching >90% conversion in 15 min. Some low-molecular-weight tailing was observed in the SEC traces when the [monomer]0 :[initiator]0 ratio was 100 (rather than 50 or 10), which suggests some early termination events occurred, but the PDIs of the final polymers were quite narrow, ranging from 1.17 to 1.23. Deprotection of the glycopolymers using a catalytic amount of methoxide occurred within minutes at room temperature but did not cleave any of the sugar units from the backbone or affect the biotin end group. The alternative to deprotection was the direct polymerization of the unprotected monomer under identical conditions, which interestingly afforded glycopolymers with narrower polydispersities (between 1.07 and 1.16) compared to the protected monomer system. The researchers also polymerized both monomers in methanol using a CuBr/L1 catalyst at 30◦ C and found that under these conditions a conversion comparable to the DMSO system was reached after 90 min and the polymerization was better controlled, with linear evolution of molecular weight with conversion and PDIs less than 1.13. Again, the unprotected monomer performed slightly better in terms of control. The biotinylated glycopolymers displayed a high affinity for the protein streptavidin. Narain was also interested in the synthesis of biotin-functionalized glycopolymers [95], and synthesized an ATRP initiator (Mn 5.1 kDa, PDI 1.07) that contained a PEG block end-functionalized with biotin. This biotin–PEG–A1 initiator was used to polymerize LAMA at 20◦ C in NMP using a CuBr/L1 catalyst system. The obtained polymers displayed PDIs less than 1.35. Binding of the biotinylated glycopolymers was investigated using a mutant streptavidin protein, which is much more conducive to biotin exchange assays than wild-type streptavidin. Fluorescein-biotin was used to first occupy all binding sites of the mutant streptavidin. Then in the presence of excess biotin-functionalized glycopolymer the fluorescent species was displaced and provided a means to quantify the degree of binding between the polymer and the protein. Unsurprisingly, the high-molecular-weight polymer (24 kDa) was sterically prohibited from accessing all four binding sites on each streptavidin, whereas the lower molecular weight species (11 and 16 kDa) were able to occupy all four binding sites. Broyer et al. developed a new procedure to modify amino acids with ATRP initiators (Fig. 1.13) [96]. A serine–A1 species was synthesized in five steps and used to initiate the ATRP of the protected glucosamine monomer (Table 1.4, entry 10) used by V´azquez-Dorbatt and Maynard [94]. After proving the robustness of the initiator for the polymerization of HEMA in MeOH at 23◦ C using CuBr/L1 at various [monomer]0 :[initiator]0 ratios, the initiator was applied to the polymerization of a methacrylic glucosamine monomer in a 85:15 methanol–water mixture. The small

49

CONTROLLED/LIVING RADICAL POLYMERIZATION

V

M

O

H N

OH V

O O

Br

V

Q

T

K

G

+

HO HO

O

O O NHAc

O

CuBr/L1 4:1 MeOH/H O 23°C

V

M

O

H N

V

V

Q

O

Fluorophore

Br n

O O

OAc AcO AcO

T

K

G

Fluorophore

O

O O NHAc

FIGURE 1.13 Fluorescent peptide–glycopolymer conjugate synthesised by ATRP from an A1-functionalized amino acid by Broyer et al. [96].

fraction of water was necessary to prevent precipitation of the polymers at higher molecular weights. Slight curvature was noted in the pseudo-first-order kinetic plot, but Mn increased linearly with conversion and the PDI of the final polymer (15.4 kDa, 84% conversion) was low at 1.19, indicating that the presence of water did not have a significant detrimental effect on the polymerization. Given these promising results, the serine-based initiator was incorporated into a model peptide containing nine amino acid residues using solid-phase peptide synthesis, and polymerization under similar conditions generated a well-defined conjugate (12.2 kDa, 93% conversion) with PDI of 1.14. Proof that the polymer was indeed conjugated to the peptide was provided by incorporating a fluorophore into the lysine (K) residue and measuring the fluorescence of the purified polymer. This important work provided a more efficient and selective method of conjugating proteins with (glyco)polymers at particular amino acids, compared to previous attempts to selectively modifying premade sequences. Vasquez-Dorbatt and Maynard performed ATRP of the same unprotected monomer (Table 1.4, entry 11) using a pyridyl disulfide-containing A1 derivative in 3:1 methanol–water at 30◦ C utilizing a CuBr/CuBr2 /L1 catalyst system [97]. Polymerization in DMSO was exceedingly rapid, reaching 90% conversion in less than 5 min, and methanol, as previously discovered [96], was not suitable due to its inability to solubilize the higher molecular weight chains. Optimization of the polymerization using A1 revealed that 3:1 methanol–water containing 1:1 CuBr/CuBr2 was most effective in controlling the polymerization. A kinetic study using the pyridyl disulfide–A1 initiator under these optimal conditions showed a linear first-order kinetic plot up to 80% conversion and PDIs less than 1.2 throughout. The polymer used for subsequent steps had Mn of 13 kDa and PDI of 1.12. Interestingly, this measured Mn was much higher than the theoretical Mn of only 3.2 kDa, a discrepancy that was also observed in the polymerization of this monomer using the biotinylated initiator [94] but not with an amino-acid-containing initiator [96]. The same initiating group is common to all three systems; therefore, the low initiator efficiency for the pyridyl disulfide species was undetermined. Some chain transfer to the pyridyl disulfide species was also observed. After purifying the polymer, the pyridyl disulfide was conjugated to thiol-terminated small inhibitory ribonucleic acid (siRNA). A dithiothreitol (DTT) solution quantitatively reversed the conjugation to release free siRNA, which is promising for the use of this conjugate system for therapeutic siRNA delivery.

50

SYNTHESIS OF GLYCOPOLYMERS

Symmetrical disulfide ATRP initiators with A1 end groups were utilized by Mizukami et al. and Kitano et al. to polymerize LAMA in 4:1 NMP–water and 2-methacryloxyethyl-d-mannopyranoside (MEMan; Table 1.4, entry 12) in methanol using CuBr/L1 at room temperature [98]. The MEMan polymerization was reasonably well controlled (PDI less than 1.5) up to high conversion, but control was poorer in 7:3 NMP–water. The disulfide-containing polymers were accumulated on surfaces covered by a monolayer of colloidal gold, and the resulting galactose- and mannose-displaying surfaces were able to reversibly associate Con A and RCA120 , respectively.

1.4.3 Reversible Addition–Fragmentation Chain Transfer Polymerization Reversible addition–fragmentation chain transfer (RAFT) polymerization is a controlled polymerization technique developed by researchers at CSIRO in 1998 [99]. Around the same time, French researchers revealed a technique referred to as macromolecular design by interchange of xanthate (MADIX), which operates via the same mechanism and can be viewed as a subset of RAFT [100]. RAFT is the most recent of the controlled polymerization techniques to garner widespread interest, primarily due to its tolerance to a wide range of reaction conditions and monomers that are difficult to polymerize in a controlled fashion using other controlled radical polymerization techniques. Glycomonomers are an important class of monomers that can be polymerized in a relatively routine manner using RAFT. In contrast to NMP and ATRP, the RAFT process is more conducive to the controlled polymerization of unprotected glycomonomers. Control is attained in a RAFT polymerization by the involvement of a thiocarbonyl thiocompound called a RAFT agent (I, Scheme 1.4). The RAFT process consists of the familiar initiation, propagation, and termination steps encountered in conventional free radical polymerization but with two superimposed equilibria controlling the growth of the polymer chains. The first equilibrium, called the pre-equilibrium, involves the addition of a propagating radical Pn • to the RAFT agent I, generating the RAFT-centred radical II. This radical can fragment in either direction; that is, revert to its original structure (and release Pn • ) or undergo ␤-scission to generate the macro-RAFT agent III and release the new radical R• . Either of the two released radicals is capable of undergoing propagation by reacting with monomer units. The pre-equilibrium ceases once every RAFT agent molecule in the system has undergone this forward fragmentation process to release its R• group and form a new oligomeric RAFT species. The second equilibrium, known as the main equilibrium, closely mirrors the preequilibrium, but in this case it is the macro-RAFT species III, which acts as the chain transfer agent. Addition of a propagating radical Pm • to III gives the RAFT-centred radical IV, which can fragment in the forward direction to release Pn • and generate another macro-RAFT species essentially the same as III (ignoring differences in chain length).

51

CONTROLLED/LIVING RADICAL POLYMERIZATION

Initiation Initiator

M ki ,1

I

I M

M kp

Pn

Pre-equilibrium and propagation

Pn

+

kadd

S R

S

Pn S

k-add

S R

kβ k-β

Pn S

Z

Z

Z

I

II

III

S

+

S

+

R

M k p

Re-initiation M k i ,2

R

M kp

R M

Pm

Main equilibrium and propagation

Pm +

S

kaddP

S Pn

k-addP

Pm S

S Pn

k-addP kaddP

Z

Z

Pm S

Pn

Z M k p

M k p III

IV

III

Termination Pm

+

Pn

kt

Dead polymer

SCHEME 1.4 RAFT mechanism.

The structure of the RAFT agent is crucial to its ability to control the polymerization of a particular monomer. RAFT agents generally fall into four classes of compounds according to the nature of their Z groups; dithioesters (Z = R ), xanthates (Z = OR ), dithiocarbamates (Z = NR R ) and trithiocarbonates (Z = SR ). The Z group determines the susceptibility of the C=S bond to radical addition, and also the lifetime of the intermediate RAFT-centred radical (II, Scheme 1.4). The choice of R group also has a pronounced effect on the performance of the RAFT agent and allows fine-tuning of its overall reactivity. The R group must be a good free radical leaving group, and the resulting radical must be capable of adding to the monomer. Detailed explanations into the appropriate choice of RAFT agent for the controlled polymerization of a particular monomer are provided elsewhere [101] and will therefore not be discussed further. Table 1.5 contains the glycomonomers in order of appearance in

52 Entry 1

2

3

Glucose

Glucose

Mannose

OH

O O O

O

O

OH OMe

O

HO O

OMe

Methyl 6-O-methacryloyl-α-Dmannoside (6-O-MAMMan)

HO HO

O

O

Methyl 6-O-methacryloyl-D-glucoside (6-O-MAMGlc)

HO HO

O

2-Methacryloxyethyl glucoside (2-MAOEGlc)

HO HO

OH

Monomer

Glycomonomers Polymerized Using RAFT

Sugar

TABLE 1.5

9:1 H2 O/EtOH

9:1 H2 O/EtOH, H2 O 86:14 D2 O/d6 -DMSO

9:1 H2 O/EtOH

H2 O 9:1 H2 O/EtOH

Solventa

70◦ C

60◦ C

70◦ C

70◦ C

70 C 70◦ C

◦

ACPA

ACPA

ACPA

ACPA

ACPA ACPA

Temperature Initiatorb

poly(2MAOEGlc)-C1

C1

C1

C1

C1 C1

RAFT Agent

103

108

103, 105, 119, 130 104

102 103

Reference

53

4

5

6

Mannose

Glucose

Galactose

HO O

O

OH

OH

O OH

O

O

O

O O

6-O-methacryloyl-1,2;3,4-di-Oisopropylidine-D-galactopyranose (MAIpGal)

O

OO

O

6-O-vinyladipoyl-D-glucopyranose (6-O-VAGlc)

HO HO

O

O

6-O-methacryloyl mannose (MAM)

HO HO

O

DMF

H2 O, MeOH DMAc

9:1 H2 O/EtOH DMAc

60◦ C

60◦ C 70◦ C

70◦ C 70◦ C

AIBN

ACPA ACPA

ACPA AIBN

C4, C5

C2, C3 C8

C1 Acetylene-C1

(continued)

110

109 116

107 123

54 Entry 7

8

Glucose

Glucose

NH

O O

O

(CH2)n

O

n = 5 10

O O

O

O

3-O-methacryloyl-1,2:5,6-di-Oisopropylidine-D-glucofuranose (MAIpGlc)

O O

O

3'-(1',2':5',6'-di-O-isopropylidine-D-glucofuranosyl)-6-methacrylamido hexanoate (n = 5) 3'-(1',2':5',6'-di-O-isopropylidine-D-glucofuranosyl)-11-methacrylamido undecanoate (n = 10)

O O

O

Monomer

Glycomonomers Polymerized Using RAFT (Continued)

Sugar

TABLE 1.5

Hexadecane/H2 O emulsion Ethyl acetate

n = 5: Dioxane n = 10: Anisole

Solventa

AIBN AIBN

75◦ C

AIBN AIBN

70◦ C

70 C 80◦ C

◦

Temperature Initiatorb

C11

C4, C5, C6

C5 C5

RAFT Agent

121

114

113

Reference

55

9

10

11

Fructose

Galactose

Glucosamine

O

O

O

O

O

O O

O

NH

O OH

N-acryloyl-D-glucosamine (AGA)

HO HO

OH

6-O-(4-vinylbenzyl)-1,2:3,4-di-Oisopropylidine-D-galactopyranose (VBIG)

O

OO

3-O-methacryloyl-1,2;5,6-di-Oisopropylidine- -D-fructopyranose (MAIpFrc)

O O

O

5:1 H2 O/EtOH H2 O 5:1 H2 O/EtOH 5:1 H2 O/EtOH

Toluene

Hexadecane/H2 O emulsion

60◦ C 65◦ C 60◦ C 60◦ C

90◦ C

70◦ C

ACPA ACPA ACPA ACPA

AIBN

AIBN

C9, C10 ␤-CD-C9 C9 C9, silica-C9

C7

C4, C5, C6

(continued)

117 118 125 127

115

114

56 12

13

14

Glucose

Glucose

Entry

OH

O O O

O

O

NH

O OH

OAc

O O O

O

2-(2',3',4',6'-tetra-O-acetyl-β-D-glucopyranosyloxy) ethyl methacrylate (AcGEMA)

AcO AcO

OAc

2-Methacrylamido glucopyranose (MAG)

HO HO

OH

2-( -D-galactosyloxy)ethyl methacrylate (GalEMA)

HO

HOOH

Monomer

Glycomonomers Polymerized Using RAFT (Continued)

Galactose

Sugar

TABLE 1.5

MeOH

9:1 DMAc/H2 O

9:1 H2 O/EtOH

Solventa

70◦ C

60◦ C

70 C

◦

AIBN

AIBN

ACPA

Temperature Initiatorb

C5

C1

C1

RAFT Agent

122

120

119, 129, 130

Reference

57

15

16

17

Galactose

Galactose

Lactose

O

O O

CHO

O

O O

O OAc AcO

O

OAc

AcO

O O

2-O-methacryloxyethyl-(2,3,4,6-tetra-O-acetyl-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-D-glucopyranoside (MAEL)

AcO

AcO OAc

6-O-acryloyl-1,2;3,4-di-O-isopropylidineα-D-galactopyranose (AIpGal)

O

OO

O

1,2:3,4-di-O-isopropylidine6-O-(2′-formyl-4′-vinylphenyl)D-galactopyranose (IVDG)

O

OO

O

O

Chloroform

␣,␣,␣Trifluorotoluene

THF

70◦ C

70◦ C

60◦ C

AIBN

AIBN

AIBN

C4

C12, PCL-C13

C6

(continued)

128

126

124

58 Entry 18

19

20

Glucose

Lactose

Galactose

OH

O O HO

OH OH H N HO O O

O

O O

6-O-acrylamido-6-deoxy-1,2:3,4-diO-isopropylidine-α-D-galactopyranose (GalAm)

O

O NH

O

2-Lactobionamidoethyl methacrylate (LAMA)

HO HO

OH

O

O

methacrylate (GAMA)

OH H N HO O

D-gluconamidoethyl

HO HO

OH

Monomer

O

Glycomonomers Polymerized Using RAFT (Continued)

Sugar

TABLE 1.5

Dioxane

5:1 H2 O/DMF

3:2 H2 O/DMF, 4:1 H2 O/MeOH

Solventa

90◦ C

60◦ C, 65◦ C

AIBN

ACPA

C16

C14, C15

C14, C15

60◦ C, 65◦ C ACPA

RAFT Agent

Temperature Initiatorb

132, 133

131

131

Reference

59

21

22

23

24

Mannose

Glucosamine

Glucosamine

Glucose

O

NH

O

NHAc

O O

NH

O

O

N H

NH

O OH O O

OH H N HO O N H

O

2-Gluconamidoethyl methacrylamide (GAEMA)

HO HO

OH

2-Deoxy-2-N-(2′-methacryloyloxyethyl) aminocarbamyl-D-glucose (GUMA)

HO HO

OH

p-Acrylamidophenyl N-acetyl-β-glucosamine

HO HO

OH

p-Acrylamidophenyl α-mannoside

HO HO

OH HO O

7:1 H2 O/DMF 7:1 H2 O/DMF 1:1 H2 O/Dioxane

3:1 H2 O/MeOH

H2 O/DMSO

H2 O/DMSO

70◦ C 70◦ C 70◦ C

70◦ C

60◦ C

60◦ C

ACPA ACPA ACPA

ACPA

AAPD

AAPD

C1 C1 C13

C1

C17

C17

(continued)

136 137 139

135

134

134

60 25

26

27

Lactose

Glucose

Mannose

OH

O O HO

OH OH H N HO O N H

O

OH H N HO O H N O

N N N

2′-(4-vinyl-[1,2,3]-triazol-1-yl)ethylO-α-D-mannopyranoside

HO HO

OH HO O

3-Gluconamidopropyl methacrylamide (GAPMA)

HO HO

OH

2-Lactobionamidoethyl methacrylamide (LAEMA)

HO

HOOH

Monomer

H2 O/MeOH

5:1 H2 O/DMF

7:1 H2 O/DMF

Solventa

60◦ C

70◦ C

70◦ C

ACPA

ACPA

ACPA

Temperature Initiatorb

C9

C1

C1, C13

RAFT Agent

14

137, 138

136 138

Reference

a EtOH = ethanol; D O = deuterium oxide; DMSO = dimethyl sulfoxide; DMAc = N,N-dimethyl acetamide; MeOH = methanol; DMF = N,N-dimethyl formamide; 2 THF = tetrahydrofuran. b ACPA = 4,4 -azobis(cyanopentanoic acid); AIBN = 2,2 -azobisisobutyronitrile; AAPD = 2,2 -azobis(2-amidinopropane)dihydrochloride.

Entry

Glycomonomers Polymerized Using RAFT (Continued)

Sugar

TABLE 1.5

61

CONTROLLED/LIVING RADICAL POLYMERIZATION

C1 S

C2 S

CN OH

S

C3 OH

S

N

C4

S

CN

O

S O

S

O

S

O

O

C8 C7

C6

C5 S

O

S

S

O

O O

S

S

O

S S

CN

S

S

S O

S O

S

O

O

C9

S

S

O S

S

S

OH

C11

S

O

O

S

S

C13

C14

S

S

S

S

S

OH

HOOC

S

S

C12 C12H25

S S

O

S

S

C15 S

S

COOH

HOOC

O

H N

S

O

O

O

S

S

C12H25

C17

C16 S

O

O

S

O

O

O

S

S

C10

S

O S

S

S

N H

OH

S O

NH

HN O

FIGURE 1.14 RAFT agents used for the synthesis of glycopolymers.

the text and their corresponding RAFT polymerization conditions. The RAFT agents and initiators used for glycopolymer synthesis are included in Figures 1.14 and 1.15, respectively. For simplicity, RAFT agents will generally be referred to by their label rather than their chemical name. Since the number of initiators is small, they will be referred to by their commonly accepted acronyms.

O

NH2

CN N

HO NC

OH

N O

4,4'Azobis(cyanopentanoic acid) (ACPA)

NC

N

N

CN

2,2'-Azoisobutyronitrile (AIBN)

HCl·HN

N

N

NH·HCl NH2

2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPD)

FIGURE 1.15 Initiators used for the RAFT polymerization of glycomonomers.

62

SYNTHESIS OF GLYCOPOLYMERS

1.4.3.1 Homopolymers and Copolymers The RAFT process is not restricted to the generation of homopolymers, although the first literature reports of glycopolymer synthesis naturally focused on single monomer systems. Well-defined random copolymers are also accessible by RAFT provided the comonomer reactivities are well matched to the RAFT agent. The “living” nature of RAFT polymerization also provides the possibility to chain extend homopolymers with another monomer to generate block copolymers. This section will focus on homopolymers and statistical/block copolymers generated without the intent of higher applications such as self-assembly or bioconjugation. The first polymerization of glycomonomers using RAFT was reported by Lowe et al. [102] in which 2-methacryloxyethyl glucoside (2-MAOEGlc; Table 1.5, entry 1) was polymerized in an aqueous system using the now commonly used RAFT agent (4-cyanopentanoic acid)-4-dithiobenzoate C1 and the water-soluble initiator 4,4 -azobis(4-cyanopentanoic acid) (ACPA) (Fig. 1.16). The solubility of the RAFT agent and the initiator were enhanced by choosing slightly basic polymerization conditions, which may have contributed to higher than expected Mn values beyond 40% conversion as a result of RAFT agent hydrolysis. Despite this, however, the low PDIs below 1.07 and the pseudo-first-order kinetics indicate that the polymerization was well controlled. No induction period was observed. Retention of the dithioester end groups was also supported by the chain extension of a poly(MAOEGlc)-C1 macro-RAFT agent (Mn = 14.2 kDa, PDI = 1.07) with 3-sulfopropyl methacrylate (SPMA), in which no low-molecular-weight tailing was observed to indicate a loss of active RAFT functionalities. However, broadening of the molecular weight (MW) distribution occurred, with the PDI rising to 1.63 accompanied by the appearance of high MW termination products in the SEC traces. A self-blocking experiment [chain extension of poly(MAOEGlc)-C1 with MAOEGlc] also resulted in a PDI over 1.5. In contrast to this, the chain extension of a poly(SPMA) macro-RAFT agent with 2-MAOEGlc gave a narrowly dispersed block copolymer with a PDI of 1.18. 1-O-methyl-␣-d-glucopyranose and 1-O-methyl-␣-d-mannopyranose were regioselectively acylated in the 6-position with vinyl methacrylate using an enzymatic approach by Albertin et al. to give the monomers 6-O-methyl-␣-d-glucopyranose (6-O-MAMGlc; Table 1.5, entry 2) and 6-O-methyl-␣-d-mannopyranose (6O-MAMMan; Table 1.5, entry 3) [103]. Stereoselective functionalization of

O O OH HO HO

ACPA, C1 H2 O, 70°C

S

NC HO

CH2

C

O

O

S n O

O

O

OH

OH HO HO

O O OH

FIGURE 1.16 Homopolymerization of 2-methacryloxyethyl glucoside (2-MAOEGlc) by Lowe et al. [102].

CONTROLLED/LIVING RADICAL POLYMERIZATION

63

unprotected sugars has been employed by several researchers in good yields without the need for protection/deprotection steps. 6-O-MAMGlc and the commercially available monomer 2-methacryloxyethyl glucoside (2-MAOEGlc; Table 1.5, entry 1) were polymerized utilizing C1 as RAFT agent and ACPA as initiator, but the addition of 10% ethanol as cosolvent was preferred to aid the dissolution of these two species, rather than risking C1 hydrolysis by adopting the basic conditions chosen by Lowe et al. [102]. Indeed, a direct comparison between aqueous RAFT using carbonate–bicarbonate versus 10% ethanol to dissolve C1 found that the added base resulted in long inhibition periods, significant retardation, bimodal molecular weight distributions, and loss of control at moderate conversions, all of which are attributed to hydrolysis of the RAFT agent. In contrast, control was maintained in the ethanol system up to high conversion [104]. Pseudo-first-order polymerization kinetics for the polymerizations of 6-OMAMGlc and 2-MAOEGlc were observed after a short induction period, indicating that the radical concentration was constant throughout. In both cases, the molecular weight increased linearly with conversion and PDIs remained below 1.12 throughout. Two resulting macro-RAFT agents were chain extended with 2-MAOEGlc and 6-O-MAMMan, respectively, to give the block copolymers poly(6-O-MAMGlc)-bpoly(2-MAOEGlc) and poly(2-MAOEGlc)-b-poly(6-O-MAMMan). Increasing the C1 concentration in the polymerization of MAOEGlc resulted in a marked retardation in the polymerization rate [105], a phenomenon which is common in RAFT polymerization using dithioesters and has been tentatively, although not conclusively, attributed to slow reinitiation in the RAFT pre-equilibrium [106]. A similar effect was observed by the same researchers in the homopolymerization of 6-O-methacryloyl mannose (MAM; Table 1.5, entry 4) [107]. Chain extension of poly(2-MAOEGlc) with 2-hydroxyethyl methacrylate (HEMA) gave well-defined hydrophilic-hydrophilic block copolymers (PDI of 1.2), although the chain extension was not quantitative because the first block was polymerized to high conversion (98%), which encouraged loss of RAFT end groups through termination in the latter stages when the monomer was depleted [105]. A detailed investigation into the kinetics of 6-O-MAMGlc polymerization using nuclear magnetic resonance (NMR) revealed that the initial non-steady-state (induction) period previously observed [103] was inversely proportional to the [C1]0 :[initiator]0 ratio [108]; that is, a higher initiator concentration (keeping the RAFT agent concentration unaltered) reduces the induction period. Interestingly, replacing C1 with a poly(6-O-MAMGlc)-C1 macroRAFT agent did not completely eliminate this induction period. The vinyl adipoyl monomer 6-O-vinyladipoyl-d-glucopyranose (6-O-VAGlc; Table 1.5, entry 5) was synthesized in a similar enzymatic fashion by Albertin et al. [109] and was polymerized at 60◦ C using a dithiocarbamate RAFT agent C2 in water and a xanthate RAFT agent (or more specifically a MADIX agent) C3 in methanol. Vinyl esters are relatively unreactive monomers whose propagating radicals are, therefore, very reactive, and as a result, controlled polymerization of these monomers is difficult. The two aforementioned RAFT agents are suitable for this purpose because both of their Z groups are electron donating and therefore have a destabilizing effect on the intermediate RAFT radical in the pre-equilibrium (species II in Scheme 1.4).

64

SYNTHESIS OF GLYCOPOLYMERS

The destabilizing nature of these Z groups encourages forward fragmentation of the RAFT-centered radical, a condition that is necessary for controlled polymerization. Excellent control was observed for both homopolymerizations, with the PDI of 1.19 attained using C2, and 1.10 using C3; however, the conversions were much lower than the control experiments without RAFT agent, particularly in the case of C2. No investigation into the kinetics was performed. Lowe and Wang polymerized the protected monomer 3-O-methacryloyl-1,2;3,4di-O-isopropylidine-d-galactopyranose (MAIpGal; Table 1.5, entry 6) using cumyl dithiobenzoate C4 and 1-cyano-1-methylethyl dithiobenzoate C5 as RAFT agents in DMF at 70◦ C [110]. The C4-mediated polymerization exhibited a 50-min induction period before proceeding with first-order kinetics. The induction period is common in C4 systems and is attributed to slow fragmentation of the intermediate RAFT-centered radical. Other explanations have also been proposed, such as an initialization period [111] and the influence of C4 impurities [112], but none have been unequivocally verified. Number-average molecular weights increased linearly with conversion, and although a discrepancy was noted between theoretical and SEC-determined Mn values, the polymerization was well controlled (PDIs below 1.20). A series of poly(MAIpGal) macro-RAFT agents were chain extended with (2-dimethylamino)ethyl methacrylate (DMAEMA) with good control, and deprotection of the isopropylidine groups using aqueous formic acid gave well-defined double-hydrophilic block copolymers. Methacrylamide variants of MAIpGlc with C5 and C10 spacers between the vinyl ¨ urek et al. using C5 and sugar groups (Table 1.5, entry 7) were polymerized by Ozy¨ [113]. Control was reasonable at best and declined beyond 50% conversion. Very broad molecular weight distributions (over 1.5) were obtained when a well-defined poly(NiPAAm) macro-RAFT agent was chain extended with the sugar monomers. Despite this lack of optimization, the thermoresponsive properties of a variety of random and block copolymers were investigated and were found to depend, among other parameters, on the spacer length of the sugar monomers. 3-O-methacryloyl-1,2:5,6-di-O-isopropylidine-d-glucofuranose (MAIpGlc; Table 1.5, entry 8) and the fructose-based monomer 3-O-methacryloyl-1,2;5,6-di-Oisopropylidine-␣-d-fructopyranose (MAIpFrc; Table 1.5, entry 9) were polymerized by Al-Bagoury et al. in a miniemulsion system using the three RAFT agents 1phenylethyl dithiobenzoate C6, C5, and C4 [114]. C6 was ineffective in controlling the polymerizations, giving significant retardation, much higher than expected Mn ’s, and broad PDIs due to its low chain transfer constant to methacrylates. Similar effects were encountered using C5, which were in this case partly attributed to easy escape of the small and highly polar 2-cyanoprop-2-yl free radical into the water continuous phase, which reduced the number of radicals in the system. In contrast, C4 allowed the generation of well-defined homopolymers (PDIs of 1.10–1.25) and the number of particles Np present throughout the polymerization followed a profile typical of a controlled miniemulsion process. Higher C4 concentrations favored better control. Interestingly, the polymerization of MAIpFrc was significantly slower than that of MAIpGlc, which was attributed to greater steric hindrance around the vinyl group in the pyranose ring of MAIpFrc compared to the furanose ring of MAIpGlc.

CONTROLLED/LIVING RADICAL POLYMERIZATION

65

Wang et al. polymerized 6-O-(4-vinylbenzyl)-1,2:3,4-di-O-isopropylidine-dgalactopyranose (VBIG; Table 1.5, entry 10) using benzyl dithiobenzoate C7 in a well-controlled manner to give optically active homopolymers (courtesy of the chiral pendant sugar groups) [115]. The polymers were useful for chiral recognition of several racemic compounds.

1.4.3.2 Stars The scope of the RAFT technique was extended by the development of multifunctional RAFT agents that allow the synthesis of star polymers. Both the R group and the Z group approaches have been applied to star glycopolymer synthesis and are detailed below. Bernard et al. synthesized glycostars by polymerizing 6-O-VAGlc (Table 1.5, entry 5) in N,N-dimethylacetamide (DMAc) at 70◦ C using a tetrafunctional xanthate RAFT agent C8 [116]. The R-group approach was adopted in this case, which means that the star acts as the R group in the RAFT process, and consequently the polymer chains grow from the core outward with the dithioester group residing at the terminus of each arm. The alternative Z-group approach has each RAFT group attached via its Z group to the star. In this case, the radicals propagate as linear chains in solution and must return to the dithioester groups, which remain at the core, in order to undergo reversible chain transfer. Both approaches have their advantages and disadvantages, but in this case the R-group approach gave unexpectedly low conversions, which did not exceed a maximum of 50% despite a continuing radical flux. Linear increase in molecular weight was observed with conversion, but the molecular weights were systematically higher than expected, which was attributed to competing side reactions that were not specifically identified by the authors. N-acryloyl glucosamine (AGA; Table 1.5, entry 11) was polymerized by Bernard et al. using mono- and trifunctional trithiocarbonate RAFT agents C9 and C10 in 5:1 H2 O/EtOH [117] and the polymerizations in this case were well controlled and reached high conversions in less than 7 h. Synthesis of the linear block copolymer poly(AGA)-b-poly(NiPAAm) was successful but was accompanied by the presence of some low-molecular-weight material that suggested incomplete chain extension. The initial attempt at star glycopolymer synthesis was performed using the Z-group approach in polar aprotic solvents to ensure solubility of both monomer and RAFT agent, but despite achieving good solubility, the control was poor. The problem was overcome by first polymerizing 2-hydroxyethyl acrylate (HEA) to give a trifunctional macro-RAFT agent whose short poly(HEA) blocks permitted dissolution in the H2 O/EtOH cosolvent system used for the subsequent sugar polymerization. The molecular weight increased with conversion, but not linearly, and polydispersities were low (1.3–1.6) but not comparable to those of the linear copolymers. Steric congestion around the RAFT groups at higher conversion is blamed for this loss of control. Reasonably well-defined poly(AGA) glycostars were synthesized similarly by Zhang and Stenzel using ␤-cyclodextrin (␤-CD) with seven C9 functionalities, but chain extension using NiPAAm proved unsuccessful [118]. 1.4.3.3 Self-Assembly The ability of the RAFT technique to generate block copolymers presents the opportunity to synthesize copolymers with blocks of

66

SYNTHESIS OF GLYCOPOLYMERS

disparate character, which can often self-assemble in an appropriate solvent to give higher order structures such as micelles or vesicles. In an aqueous system, the hydrophobic core of the micelles can be designed to encapsulate a therapeutic species such as hydrophobic drug or gene. Glycopolymers are interesting candidates for the hydrophilic shell because their biological activity can facilitate targeted delivery of the drug to particular cells. As an interesting anomaly, the final example in this section demonstrates the self-assembly of a sugar-containing homopolymer rather than a block copolymer. Cameron et al. synthesized block copolymers that self-assembled in aqueous solution into wormlike micelles capable of encapsulating a hydrophobic dye. The hydrophilic block was synthesized from 2-(␤-d-galactosyloxy)ethyl methacrylate (GalEMA; Table 1.5, entry 12) and the hydrophobic block from n-butyl acrylate (BA) with high blocking efficiency and PDIs less than 1.2 [119]. A block copolymer synthesized by Pearson et al. also exhibited a tendency to form rods rather than spherical micelles in solution as the length of the hydrophobic block was increased. Homopolymerization of 2-methacrylamido glucopyranose (MAG; Table 1.5, entry 13) to generate the hydrophilic block was well controlled by C1 [120]. The polymerization of 3-O-methacryloyl-1,2:5,6-di-O-isopropylidine-d-glucofuranose (MAIpGlc; Table 1.5, entry 8) using cumyl phenyl dithioacetate C11 RAFT agent by Ramiah et al. showed an apparent lack of control in the early stages of the polymerization, after which the PDI decreased from 1.5 down to 1.16 at higher conversion [121]. This feature of C1-mediated polymerization of (meth)acrylates is attributed to slow fragmentation of the RAFT-centered radicals in the pre-equilibrium (II, Scheme 1.4). A conventional free radical polymerization mechanism therefore dominates at low conversion until all of the RAFT agent species have undergone fragmentation, after which the polymerization proceeds in a controlled manner and the PDI declines. Chain extension with styrene or methyl acrylate generated amphiphilic block copolymers that formed spherical micelles in aqueous solution after deprotection of the isopropylidine groups. Paspakaris and Alexander polymerized acetyl-protected 2glucosyloxyethyl methacrylate (AcGEMA; Table 1.5, entry 14) using C5 and, after deprotection, chain extended with diethyleneglycol methacrylate (DEGMA) to give a thermosensitive block copolymer [122]. In aqueous solution the polymer chains formed vesicles whose diameters decreased from 500 to 300 nm when the temperature of the system exceeded 37◦ C. Above this temperature, the thermoresponsive poly(DEGMA) block becomes hydrophobic and therefore the PDEGMA core of the vesicles collapses. These thermoresponsive vesicles were used to investigate polyvalent binding events of Con A and mutant Escherichia coli, which expresses specific receptors for glucose and mannose. A block copolymer of vinyl acetate (VAc) and 6-O-methacryloyl mannose (MAM; Table 1.5, entry 4) was synthesized by Ting et al. [123], but a macromolecular click reaction was used rather than chain extension of a macro-RAFT agent because the large difference in monomer reactivities precludes their union in this manner; the success of the RAFT process relies on careful selection of R and Z groups according to the reactivity of the propagating species, and in this case the reactivities of the two monomers are too different to permit a chain extension approach. MAM was

67

CONTROLLED/LIVING RADICAL POLYMERIZATION S

S C6, AIBN

CH2

CH

THF, 60°C CHO OO

Deprotection

O

CH2

S n CHO

HO O O

O HO

O O

CH

CHO OO

O O

S n

O

O

OH OH

FIGURE 1.17 Polymerization of 1,2:3,4-di-O-isopropylidine-6-O-(2 -formyl-4 -vinylphenyl)-d-galactopyranose by Xiao et al. [124].

polymerized using a C1-based RAFT agent containing a silyl-protected alkyne to give a well-defined glycopolymer (Mn = 7.6 kDa, PDI = 1.11), which, after deprotection of the silyl group, was reacted with an azide-functionalized poly(VAc) for 48 h in DMAc using a 1,8-diaza[5,4,0]bicycloundec-7-ene and CuI catalyst system. SEC analysis did indicate that the desired block copolymer was formed, but the PDI was inexplicably broad (1.48) due to the presence of unreacted homopolymers. A new glycomonomer containing an aldehyde functionality was developed by Xiao et al. (Fig. 1.17) [124]. Polymerization of 1,2:3,4-di-O-isopropylidine-6-O(2 -formyl-4 -vinylphenyl)-d-galactopyranose (IVDG; Table 1.5, entry 15) was performed at 60◦ C in tetrahydrofuran (THF) using C6. The Mn increased linearly with conversion and PDIs remained less than 1.10 throughout, highlighting the versatility of the RAFT process in accommodating monomers with various functional groups. Removal of the isoproylidine groups using formic acid gave an amphiphilic homopolymer that self-assembled in aqueous medium to give discrete spherical micelles of uniform size. Aldehyde groups can react with primary amines to form a Schiff base linkage under mild conditions, and were thereby used in this system to conjugate bovine serum albumin (BSA), a model protein, to the micelles.

1.4.3.4 Crosslinked Micelles Researchers from the Centre for Advanced Macromolecular Design (CAMD) have published several reports of crosslinked micelles made from glycopolymer-containing block copolymers. Zhang et al. [125] synthesized poly(AGA)-b-poly(NiPAAm) block copolymers as reported previously [117] and crosslinked the core of the resulting micelles by polymerizing with 3,9-divinyl-2,3,8,10-tetraoxaspiro[5.5]undecane (an acid-degradable crosslinker). Crosslinking in this case involved chain extension of the assembled block copolymers with a hydrophobic difunctional species that was encapsulated in the micelle core (where the RAFT end groups also resided). The process was essentially an emulsion polymerization with radicals provided by a water-soluble initiator. No gelation occurred due to successful mediation of the polymerization by the RAFT agent, giving stable core–shell structures that readily decomposed under acidic conditions. A similar approach using 1,6-hexanediol diacrylate also gave stabilized micelles [118]. A macro-RAFT agent synthesized from a poly(lactide) (PLA) was used to chain extend 6-O-acryloyl-1,2;3,4-di-O-isopropylidine-␣-d-galactopyranose (AIpGal;

68

SYNTHESIS OF GLYCOPOLYMERS

O O

O

O

O

O C13 SnOct2, 140°C

O

HO O

S

O m

S

AlpGal, AIBN, 70°C α,α,α-trifluorotoluene

S

O

HO O

S

O

S S

m

n

OO

O O

O a) Deprotection b) Self-assembly c) Crosslinking d) Core removal

O

O

Hollow sugar balls

FIGURE 1.18 Synthesis of hollow sugar balls by Ting et al. [126].

Table 1.5, entry 16) by Ting et al. (Fig. 1.18) [126]. Suitable conditions were found by polymerizing AIpGal with C12 in ␣,␣,␣-trifluorotoluene, after DMAc and DMSO proved ineffective. Polymerization using the PLA-C13 macro-RAFT agent proceeded at a higher rate than the preceding RAFT experiment under the same conditions. A higher macro-RAFT concentration reduced the polymerization rate, which is attributed to lower initiator efficiency due to the higher viscosity (rather than slow fragmentation of the intermediate RAFT radicals, since this is not usually observed with trithiocarbonates). After deprotection of the sugar, the amphiphilic block copolymer self-assembled into micelles, which were crosslinked with 1,6-hexandiol diacrylate at the interface between the two blocks (since the RAFT functionality resided there). Removal of the PLA core using hexylamine afforded hollow galactose-bearing nanocages.

1.4.3.5 Surface and Particle Modification Various strategies have been successfully employed to generate surfaces and particles functionalized with welldefined glycopolymers. The majority focus on attachment of premade glycopolymers via various reactive groups installed at the chain end or along the backbone, but the first report mentioned in this section details direct RAFT polymerization from a surface. Stenzel et al. synthesized glycopolymer brushes by attaching a trithiocarbonate RAFT agent to a silica substrate and polymerizing NiPAAm and N-acryloyl glucosamine (AGA; Table 1.5, entry 11) in a sequential fashion (Fig. 1.19) [127]. The trithiocarbonate RAFT agent C9 was affixed using the Z-group approach, which

Si O Si O Si Si O

O N H

S S

O

Si O Si O Si Si O

AGA, ACPA, C9 5:1 H2O:EtOH 60°C

S

N H

S S

S

C

CH2 m

O

NH

OH

O

HO HO OH NiPAAm, ACPA, C9 1:1 H2O:DMSO 60°C

Si O Si O Si Si O

O N H

S S

S

C

CH2

C

CH2

n HN

O

O

m NH

OH

O

HO HO OH

FIGURE 1.19 Grafting of glycopolymer from a silica surface by Stenzel et al. [127].

CONTROLLED/LIVING RADICAL POLYMERIZATION

69

means the trithiocarbonate group remained between the growing polymer chain and the silica substrate. The polymerization was performed by submerging the RAFTfunctionalized silica in a 5:1 H2 O/EtOH solution of AGA, ACPA, and free C9. The use of free RAFT agent in solution suppresses termination since the propagating radicals are not attached to the surface in the Z-group approach and are more likely to undergo combination rather than reversible chain transfer if the only RAFT agent in the system is confined to the surface. Both the molecular weight of the free polymer and the corresponding brush thickness both increased with conversion, suggesting a controlled polymerization process. Surprisingly, chain extension using NiPAAm showed none of the steric hindrance problems that plagued previous chain extensions of star macro-RAFT agents using the Z-group approach. Success in this case is attributed to entrapment of the growing radicals close to the surface, which meant they were always in close proximity to the trithiocarbonate controlling agent (which resides between the surface and the growing chains). A radical approach was used by Guo et al. to attach a polymer synthesized from 2-O-methacryloyloxyethyl-(2,3,4,6-tetra-O-acetyl-␤-d-galactopyranosyl)-(14)-2,3,6-tri-O-acetyl-␤-d-glucopyranoside (MAEL; Table 1.5, entry 17) to a vinylfunctionalized silica surface [128]. Polymerization of the protected monomer was conducted using C4 in chloroform, which is an unusual choice of solvent for controlled polymerization because of its relatively high chain transfer constant. The final PDIs were still low (1.07–1.34), but the measured molecular weights were significantly higher than expected. More judicious solvent choice may have improved the level of control. Glycosylated nanoparticles were synthesized from GalEMA (Table 1.5, entry 12) by Spain et al. in an aqueous system containing 20% ethanol using C1 at 70◦ C [129]. Despite approaching complete conversion after 2 h, the polymerization showed all indications of well-controlled living polymerization. The dithiocarbonate end group of the final polymer (Mn = 24.1 kDa, PDI = 1.09) was reduced to a thiol by NaBH4 in the presence of HAu(III)Cl4 , which was simultaneously reduced to Au(0), giving glycopolymer-stabilized gold nanoparticles (AuNPs). Thiols are well known for their strong binding to gold. The biological activity of the galactose groups on the particles was demonstrated by their ability to agglomerate peanut agglutinincoated beads. As an aside, the same polymer was included in a small library of polymers containing peptide and vinyl-derived backbones that were investigated for their antifreeze abilities [130]. The glycopolymer was found to have a small but significant inhibiting effect on crystallization. Several other reports of glyconanoparticles prepared in a similar manner have emerged since that of Spain et al. [129] was published. Trithiocarbonate-mediated polymerization of d-gluconamidoethyl methacrylate (GAMA; Table 1.5, entry 18) and 2-lactobionamidoethyl methacrylate (LAMA; Table 1.5, entry 19) gave glycopolymers that were reduced in the presence of the thiol (denoted –SH) species biotin–PEG–SH and Au(III) to give AuNPs stabilized with glycopolymer and biotinPEG. The particles were bioconjugated to streptavidin [131]. Random copolymers of 6-O-acrylamido-6-deoxy-1,2:3,4-di-O-isoproylidine-␣-d-galactopyranose (GalAm; Table 1.5, entry 20) and N-acryloylmorpholine (NAM) made using the biotinlabeled RAFT agent C16 also gave glycoparticles that could bind streptavidin [132].

70

SYNTHESIS OF GLYCOPOLYMERS

Compositional drift during the random copolymerization saw an increasing proportion of the galactose monomer incorporated in the latter stages of the polymerization [133]. AuNPs with random copolymers containing p-acrylamidophenyl ␣-mannoside (Table 1.5, entry 21) or p-acrylamidophenyl N-acetyl-␤-glucosamine (Table 1.5, entry 22) (copolymerized with acrylamide) showed strong, specific molecular recognition of lectins and bacterium [134]. A homopolymer of 2-deoxy-2-N(2 -methacryloyloxyethyl)aminocarbamyl-d-glucose (GUMA; Table 1.5, entry 23) was synthesized by RAFT using C1 after a failed attempt using ATRP due to the presence of the urea group. The resulting RAFT polymer (PDI of 1.17) was reduced and attached to a solid substrate coated in AuNPs to give a surface with excellent resistance to nonspecific protein adsorption [135]. Deng et al. polymerized the two monomers 2-gluconamidoethyl methacrylamide (GAEMA; Table 1.5, entry 24) and 2-lactobionamidoethyl methacrylamide (LAEMA; Table 1.5, entry 25) in 7:1 H2 O/DMF at 70◦ C using C1 and S,S -bis(␣,␣ -dimethyl-␣ -acetic acid)trithiocarbonate C14 RAFT agents. Surprisingly, the polymerization of the more bulky LAEMA using C1 was approximately twice as fast as that of GAEMA. A long inhibition period was observed for both. Much better control was attained using the dithioester C1 rather than the trithiocarbonate C14. Macro-RAFT agents synthesized from primary amine-containing monomers were chain extended with GAEMA, resulting in some broadening in the molecular weight distributions. Biotin was attached to the amine groups, and AuNPs stabilized with the resulting block copolymers specifically recognized both streptavidin and RCA120 lectin [136]. In a separate publication the same group found the polymerization of the slightly bulkier 3gluconamidopropyl methacylamide (GAPMA; Table 1.5, entry 26) to be slower than that of GAEMA. In this report a second primary amine-containing block was used to condense plasmid deoxyribonucleic acid (DNA) [137], and in a further publication the same poly(amine)-b-poly(GAPMA) and the lactose equivalent poly(amine)-bpoly(LAEMA) attached to single-wall carbon nanotubes (SWNTs) displayed high biocompatibility and transfection efficiency as potential gene delivery agents [138]. Jiang et al. attached a RAFT-synthesized random copolymer containing glucose (GAEMA; Table 1.5, entry 24), primary amine, and biotin pendant groups to quantum dots (Fig. 1.20) [139]. Quantum dots are semiconductor nanocrystals whose dimensions impart unique optical properties useful for biomedical imaging applications.

+

+ O O

O

O O

O

ACPA, C14 HO 70°C

HOOC

CH

C co CH

OH HO HO

O NH O

S

C n O

O

S

COOH

S

O

O

OH

OH HO

C co CH

O

NH

NH ·HCl

HO HO

O

OH NH HO

O

NH ·HCl

NH O

H N S

H N

O N H

S

O N H

FIGURE 1.20 Statistical copolymer used by Jiang et al. to functionalize quantum dots [139].

RING-OPENING POLYMERIZATION

71

The three monomers were copolymerized using C14 in an aqueous system to give a well-defined statistical copolymer with Mn = 12.9 kDa and PDI = 1.19. Reaction of the primary amine groups in the polymer with activated ester groups on the quantum dot surface had no effect on the physical properties of the quantum dots but gave water-soluble particles with the ability to recognize streptavidin and with improved biocompatibility compared to the naked particles.

1.5 RING-OPENING POLYMERIZATION Ring-opening polymerization (ROP) of cyclic monomers such as cyclic ethers, acetals, amides (lactams), esters (lactones), and siloxanes results in the formation of similar products as step polymerization products, but usually with better control over molecular weight and smaller molecular weight distributions. Ring sizes commonly used are 3-, 4-, 5-, 7-, and 8-membered rings, while the polymerization of 6-membered rings is thermodynamically unfavorable. Ring-opening polymerization can be initiated with anionic and cationic species including strong protic acids and Lewis acids in conjunction with water for cationic polymerization and alkali metals, metal alkoxides, metal complexes and others for the anionic process (Scheme 1.5). Many ROPs proceed as living polymerizations with the molecular weight increasing with conversion. However, significant chain transfer reaction can be present especially in cationic ROPs. The synthesis of complex architectures such as star and block copolymers is feasible [140]. Two reports on the synthesis of glycopolymers using ROP emerged simultaneously in 1985 when Good and Schuerch [141] and Uryu et al. [142] used the anhydride form of various sugars (Table 1.6, entries 1–3) to yield polymers of up to 11 kDa

Anionic

X R R

X

R

X * n

Cationic

X

R X

X

R R X

X

R

X * n

SCHEME 1.5 Mechanism of anionic and cationic ring-opening polymerization.

72 1

2

3

4

Galactose

Glucose

Glucose

Entry

OBz

OOBz

BzO

O

OR

OR

O OR R= p-Br C6H4CH2R= Bz R= p-CH3C6H4CH2-

OAc

O O

HN O

O

O

O-(tetra-O-acetyl-β-D-glucopyranosyl)L-serine N-carboxyanhydride

AcO AcO

OAc

1,3-anhydro-2,4,6-tri-O-alkylβ-D-glucopyranose

O

1,4-Anhydro-2,3,6-tri-O-benzylβ-D-galactopyranose

BzO

BzO O

1,4-Anhydro-2,3,6-tri-O-benzylα-D-glucopyranose

BzO

O

Monomer

Dioxane CH2 Cl2 Acetonitrile

CH2 Cl2 Toluene Benzene

CH2 Cl2

CH2 Cl2

Solvent

Glycomonomers Synthesized with Ring-Opening Polymerization

Glucose

Carbohydrate

TABLE 1.6

25◦ C

Et3 N n-HexNH2 t-BuNH2 Polyoxaline-NH2 p-Vinylbenzylamine

PF5 SbCl5 BF3 ·O(C2 H5 )2 (CF3 SO2 )2 O (C6 H5 )3 CCl

PF5 SbCl5 BF3 ·O(C2 H5 )2

−78◦ C – + 0◦ C

−78◦ C – + 50◦ C

PF5 SbCl5 BF3 ·O(C2 H5 )2 SnCl4 (CF3 SO2 )2 O (i-Bu)3 Al-H2 O

Initiator

−78◦ C – +30◦ C

Temperature

144–146, 149

141

142

142

Reference

RING-OPENING POLYMERIZATION

(a)

O

73

OH

O OR

OR

O

HO

OH O

OR

n

(b)

O

OAc AcO AcO

HN

O O OAc

O

O R-NH2

O

R

N H

H N

H n

O

O AcO

OAc

AcO AcO

FIGURE 1.21 Ring-opening polymerization of glycomonomer: (a) anhydride [141] and (b) N-carboxyanhydride [145].

(Fig. 1.21). Depending on the initiator, stereoregular glycopolymers were obtained as evidenced by optical rotation and NMR. Glycomonomers based on N-carboxyanhydrides were first reported by R¨ude et al. [143] but not until the pioneering work of Okada and co-workers were these molecules employed to design complex glycopolymer architectures (Fig. 1.21) [144]. Welldefined homopolymers were obtained by initiating the polymerization with primary amines (PDI of 1.1) while tertiary amines led to broader molecular weight distributions. The amino end-functionality of the product was then utilized to generate block copolymers in a subsequent step [145]. The initiating species for the polymerization—primary amines—provide an avenue to broaden the array of available structures. Amino-terminated polyoxazolines were used as macroinitiators to initiate the polymerization of O-(tetra-O-acetyl-␤-dglucopyranosyl)-l-serine N-carboxyanhydride (Table 1.6, entry 4), resulting in block copolymers with molecular weights which were in good agreement with the theoretical values albeit with broader molecular weight distributions (PDIs of 1.2–1.6) [146]. A similar block copolymer was created by joining two functional homopolymers, polyoxazoline generated via cationic polymerization and poly(O-(tetra-O-acetyl-␤d-glucopyranosyl)-l-serine N-carboxyanhydride) with an amino end-functionality obtained via living anionic ring-opening polymerization. Polyoxazoline was added to the anionic ring-opening polymerization, terminating the polymerization, thus yielding block copolymers with PDIs less than 1.1 [147]. Two avenues were investigated to prepare macromonomers based on carbohydrates in combination with ring-opening polymerization. Comb polymers with glucose functionalities at the end of each branch were generated from N-acetyl-d-glucosamine. The sugar molecule was converted into oxazoline, which then acted as the initiated species in the ring-opening polymerization of various 2-oxazolines (Fig. 1.22). Termination with acrylate anions and the following radical copolymerization with styrene resulted in comb polymers of molecular weights of up to 45,000 g mol−1

74

SYNTHESIS OF GLYCOPOLYMERS

OAc

OAc O

AcO AcO

N

N

MeOTf O

O

R

O-

O

R

O

O

O

AcO AcO Me

N

N O

n

O

Me

Me

FIGURE 1.22 Synthesis of macromonomers with glucose endfunctionality [148].

with a polystyrene backbone and poly(oxazoline) braches with molecular weights of 1850 g mol−1 [148]. An elegant and simple method for glycol macromonomers was developed by using p-vinylbenzylamine as the initiator for the ring-opening polymerization of O-(tetra-O-acetyl-␤-d-glucopyranosyl)-l-serine N-carboxyanhydride (Table 1.6, entry 4). Subsequent polymerization of the macromonomer with acrylamide lead to comb polymers with high activity when interacting with wheat germ agglutinin [149].

1.6 IONIC CHAIN POLYMERIZATION 1.6.1 Anionic Chain Polymerization Anionic polymerization is initiated with strong nucleophiles such as alkyl or naphthalenide anions. To date, anionic polymerization is the only polymerization method that is truly living, providing polymers with extremely narrow molecular weight distributions while termination and chain transfer reactions are usually absent. However, this technique requires stringent reaction conditions and the choice of monomers is limited since competing reaction with other functional groups need to be absent to ensure living behavior (Scheme 1.6) [150]. It is therefore not surprising that the reports on glycopolymers using living anionic polymerization are limited. Hirao and co-workers achieved well-defined glyco-homo and block copolymers with polydispersity indices as low as 1.04. Prerequisite, however, was the careful design of the structure of the glycomonomers, which were all based on styrene derivatives. Styrene with acetyl-protected ␣-d-galactopyranose, ␤-d-fructopyranose, and ␤-l-sorbofuranose in the meta position (Table 1.7, entries 1–6) underwent living anionic polymerization affording molecular weights close to predicted values. In contrast, para-substituted products resulted in no polymerization. This was explained by an 1,6-elimination step forming a biradical intermediate [151]. The hypothesis was confirmed by synthesizing a para-substituted glycomonomer with a hydrocarbon spacer between styrene and carbohydrate (Table 1.7, entry 7), which is n M+Alkyl -

Alkyl CH2 CH R

R

R

H+ Alkyl CH2 CH CH2 CH R R n

Alkyl CH2 CH H R n+1

SCHEME 1.6 Mechanism of anionic living polymerization.

75

1

2

Glucose

Entry

O

O O

O

O

O O

O

m-(1,2:5,6-Di-O-cyclohexyli dene-αD-glucofuranose-3-oxy-methyl)styrene

O O

m-(1,2:5,6-Di-O-isopropylidene-αD-glucofuranose-3-oxymethyl)styrene

O O

Monomer

Glycomonomers Synthesized via Living Anionic or Cationic Polymerization

Glucose

Carbohydrate

TABLE 1.7

THF

THF

Solvent

−78◦ C

−78 C

◦

Temperature

s-BuLi

s-BuLi

Initiator

(continued)

151

151

Reference

76 3

4

5

Fructose

Sorbose

Entry

O

O O

O

OO

O

O

O

O O

m-(2,3:4,6-Di-O-isopropylidene-αL-sorbofuranose-1-oxy-methyl)styrene

O

O

O

m-(1,2:4,5-Di-O-isopropylidene-αD-fructopyranose-3-oxymethyl)styrene

O

m-(1,2:3,4-Di-O-isopropylideneD-galactopyranose-6-oxy-methyl)styrene

O

OO

Monomer

THF

THF

THF

Solvent

Glycomonomers Synthesized via Living Anionic or Cationic Polymerization (Continued)

Galactose

Carbohydrate

TABLE 1.7

−78◦ C

s-BuLi

s-BuLi

s-BuLi

−78◦ C

−78◦ C

Initiator

Temperature

151

151

151

Reference

77

6

7

Glucose

Glucose

O

O O

O

O

OO n O

n= 3, 11

m-(1,2:5,6-Di-O-isopropylideneα-D-glucofuranose-3oxypropyl)styrene n=3

O O

m-(1,2:5,6-Di-O-isopropy lideneα-D-glucofuranose-3oxyundecyl)styrene n=11

p-(1,2:5,6-Di-O-isopropylidene-αD-glucofuranose-3-oxymethyl)styrene

O O

THF

THF

−78◦ C

−78◦ C

s-BuLi

s-BuLi potassium naphthalate

(continued)

152

151

78 8

9

Glucosamine

Entry

O O

O O

O

O

O O

O

O O

O

O

N

O O O

O

3,4,6-tri-O-acetyl-2-deoxy-2phthalimide-β-D-glucopyranoside

O

O

O

1-O-(vinyloxy)ethyl-2,3,4,6-tetra-Oacetyl-β-D-glucopyranoside

O

O

Monomer

Toluene

Toluene

Solvent

Glycomonomers Synthesized via Living Anionic or Cationic Polymerization (Continued)

Glucose

Carbohydrate

TABLE 1.7

−15◦ C or 0◦ C

−15 C

◦

Temperature

HCl/ZnI2 or TFA/EtAlCl2 (dioxane)

HCl/ZnI2

Initiator

154, 156

153

Reference

10

11

Glucose

Galactose

79

O O

O

O

O

O O

1,2:3,4-di-O-isopropylidene-6-O(2-vinyloxy ethyl)-D-galactopyranose

O

O

O

3-O-(vinyloxy)ethyl-1,2:5,6-di-Oisopropylidene-D-glucofuranose

O O

O

O

−20◦ C

2. −30◦ C

2. Benzene

Toluene

1. −40◦ C

1. Toluene

CH3 CHI(OEt)/ ZnCl2

1. CH3 CH (OiBu)Cl/ZnI2 2. PS-I/ZnCl2

159

158

156

80

SYNTHESIS OF GLYCOPOLYMERS

incapable of elimination and therefore anionic polymerization proceeded according to the expected living behavior [152].

1.6.2 Cationic Chain Polymerization Cationic polymerization can be initiated using protic acids, Lewis acids, halonium ions, or oniom salts in combination with photoinitiation. While cationic polymerization is closely related to anionic chain polymerization, chain transfer and termination reactions are frequently observed. Side reactions such as ␤-proton transfer, combination with counterion, and chain transfer to polymer limit the growth of a terminating chain. Therefore, PDIs of up to 2 can be theoretically expected and are observed. However, fast initiation and the absence of chain transfer reactions can lead to narrow molecular weight distributions. Under specific circumstances, living cationic polymerization is operational with the molecular weight increasing with conversion (Scheme 1.7) [150]. Glycopolymers via living cationic polymerization are frequently reported. A HCl/ZnI2 initiating system was employed by Minoda et al. to achieve the living polymerization of 1-O-(vinyloxy)ethyl-2,3,4,6-tetra-O-acetyl-␤-d-glucopyranoside (Table 1.7, entry 8) [153]. The molecular weight increased linearly with conversion, although it leveled off at around 80% monomer conversion. A similar result was observed with a monomer based on glucosamine (Table 1.7, entry 9) [154]. This phenomenon was attributed to poor solubility of the resulting glycopolymers in the solvent. Attempts to improve the livingness by using different solvents at higher temperatures succumbed to broader molecular weight distributions. A solution was found by adopting a different initiation system, trifluoroacetic acid with ethylaluminum dichloride, which led to a living process up to 100% monomer conversion [154]. Subsequent work by Minoda and co-workers demonstrated that the catalyst system can moreover be employed to successfully synthesize block copolymers based on poly(isobutyl vinylether) and poly(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-␤-dglucopyranoside) (Table 1.7, entry 9) (Fig. 1.23) [155]. The specific interaction of the glycopolymer obtained after deprotection and acetylation of the amino functionality with wheat germ agglutinin (WGA) was evaluated using fluorescence spectroscopy. The block copolymers showed significantly enhanced recognition abilities compared to the homopolymer, let alone the monovalent acetyl glucosamine. A similar block copolymer, based on poly(isobutyl vinylether), was obtained using a protected glucofuranose pendant group (Table 1.7, entry 10). Well-defined block copolymers with PDIs below 1.1 were created and the number of repeating units varied between 20 and 90 [156]. Upon deprotection, amphiphilic block copolymers

n H+ R

H CH2 CH R

R

H CH2 CH CH2 CH R R n

H2O

SCHEME 1.7 Mechanism of cationic polymerization.

H CH2 CH OH R

n +1

81

IONIC CHAIN POLYMERIZATION

O O

O O

O HO

O

O

O

CF3

O

EtAlCl2 1,4-Dioxane

O O N

CF3 O

O

AlEtCl2

O O

O

O O

O

O

O

R O

N

R= carbohydrate

OO O

O

O

n

m O

O

O m

n-1

δ− O

δ+ O

O

n

O

m

a) H2NNH2*H2O b) Ac2O/MeOH

O O

O O

O

O

HO

O

HOHO

OO

O

O

O

O NH

N

FIGURE 1.23 Synthesis of poly(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimide-␤-d-glucopyranoside)-block-poly(isobutyl vinylether) via living cationic polymerization [155].

were formed while simultaneously converting glycofuranose into its pyranose form. Depending on the composition, these block copolymers were found to form various morphologies in solid state, including spheres, cylinders, and lamellas. The monomer reactivity ratios of the statistical copolymerization of these two monomers, isobutyl vinylether (IBVE) and 3-O-(vinyloxy)ethyl-1,2:5,6-di-O-isopropylidene-dglucofuranose (IpGlcVE; Table 1.7, entry 10), were calculated to be r1 (IBVE) = 1.65 and r2 (IpGlcVE) = 1.15. The solution behavior of the statistical copolymer differed substantially from that of the block copolymer. As evidenced by NMR spin-lattice relaxation times, the block copolymer was in an aggregated state in solution at low temperatures while the statistical copolymer was unrestricted in motion [157]. Combination of living anionic and living cationic polymerization resulted in amphiphilic block copolymers with polystyrene as the hydrophobic block. The anionic styrene polymerization was terminated with 3-chloropropionaldehyde diethyl acetal. Addition of trimethylsilyl iodide led to a macroinitiator for cationic polymerization (Fig. 1.24) [158].

O O OO O n -1

Cl

O O

n

I O

I-SiMe

n

O O

O

O Block copolymer

ZnCl

FIGURE 1.24 Polystyrene-block-poly(glucofuranose vinyl ether) by combination of living anionic and living cationic polymerization by Labeau et al. [158].

82

SYNTHESIS OF GLYCOPOLYMERS

Glycopolymers carrying galactose moieties (Table 1.7, entry 11) were prepared for the immobilization of DNA probes. The aldehyde functionality of the acylic form of the galactose group underwent Schiff base formation with the amino-terminated oligonucleotide, which was followed by reductive amination. A more detailed investigation was dedicated to the end group analysis of the polymer obtained during the living cationic process. The polymerization was terminated in a highly alkaline solution of aqueous KOH. As a result, polymers with aldehydes as ␻-end groups were obtained [159]. Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectroscopy analysis indeed confirmed the presence of aldehyde end-functionalities but also the presence of unsaturated products as a result of ␤-elimination. 1.7 RING-OPENING METATHESIS POLYMERIZATION (ROMP) Transition-metal coordination initiators instigate the reaction of cycloalkenes to form polymers via a ring-opening process. Metal–carbene complexes such as the molybdenum- and tungsten-based Schrock initiators and the ruthenium-based Grubbs initiators allow control over the polymerization. Grubbs initiators are considered advantageous due to their low sensitivity against air, moisture, and many functional groups. Depending on the fine structure and further metal ligands, initiators with good polymerization rates in combination with limited side reactions can be designed. In general, ROMP proceeds in a living fashion with narrow molecular weight distributions and the ability to generate block copolymers (Scheme 1.8) [160]. Kiessling and co-workers pioneered the area of glycopolymers via ROMP. In their initial work, a 7-oxobornene derivative with glucose functionalities (Table 1.8, entry 1) was polymerized in water in the presence of RuCl3 at 55◦ C. The polymer obtained with a molecular weight of 106 g mol−1 showed an efficient inhibition of erythrocyte agglutination by Con A. Inhibition doses required were 2000 times lower compared to the monomeric unit, demonstrating its multivalent effect [161]. This study was extended to compare ␣-C-glucoside, ␣-O-glucoside, ␣-C-mannoside, and ␣-O-mannoside (Table 1.8, entries 1–4). The four monomers were obtained by polymerization in water, all resulting in molecular weights of 106 g mol−1 . The ␣-Cmannoside glycopolymer (Table 1.8, entry 3) was measured to have a most efficient inhibition effect of Con-A-induced hemagglutination, followed by the polymer with ␣-O-mannoside (Table 1.8, entry 4) [162]. Concerns were raised regarding the effect of ruthenium impurities in biological applications of these polymers. The use of recycled catalyst or preformed catalyst (obtained by heating RuCl3 with a small amount of monomer) not only overcame this problem, since these are more easily removed, but also enhanced the initiation rate and polymer yield [163].

RCH M

RCH M

RCH M

RCH

M

RCH=CHCH2CH2CH2CH=M n

SCHEME 1.8 Ring-opening metathesis polymerization (ROMP).

83

1

2

Glucose

Entry

HO HO

HO HO

OH

OH

Monomer

O O O

H O

O

O

O O OH HO

H

O

O

O

HO

O O

H O

OH

H O

O

OH OH

OH

OH OH

OH

Glycomonomers Polymerized via ROMP

Glucose

Carbohydrate

TABLE 1.8

Water

Water

Solvent

55◦ C

55◦ C

RuCl3

RuCl3

Temperature Initiator

(continued)

162

161

Reference

84 3

4

Mannose

Entry

Mannose

Carbohydrate

HO HO

HO HO

OH

OH

Monomer

O OH

H O

O OH

H O

O O

O O

O

O O

O

O OH

H O

OH OH

OH OH

OH

OH

O OH

H O

TABLE 1.8 Glycomonomers Polymerized via ROMP (Continued)

Water

Water

Solvent

55◦ C

55 C

◦

RuCl3

RuCl3

Temperature Initiator

162

162

Reference

85

5

6

7

Mannose

Glucose

Galactose

O

HO

H O

OH

O

O OH

H O

OH OR

O O

O

O O

MO3SO

H O

H O

O

O

HN

O

OH

OH OH

OH

OH OH

R= H or MO3S

O

Water/ ClCH2 CH2 Cl/ DTAB

Water

Water

65◦ C

55◦ C

55◦ C

P(Cy)3 Ph Ru H Cl P(Cy)3 Cl

RuCl3

RuCl3

(continued)

164

163, 164

163

86

Glucose

Galactose

Carbohydrate

10

9

8

Entry

O3SO

O

RO HN

-

O

OH

O

OR'

OR OR

O

OH

O

HO OH

H3C

OH OR6'

MO3SO

OH OR

Monomer

O

OR6 OH

O O

HN

R= H or MO 3S

O

R=R'=H I R=R'=Ac II R=R'=CH2Ph III R=R'=SiEt3 IV R=H; R'=CPh3 V

OH

O

O

HN

TABLE 1.8 Glycomonomers Polymerized via ROMP (Continued)

R6=SO3-, R6'=H

R6= H, R 6'= SO 3-

O

CH2 Cl2 Benzene and solvent mixtures

Water/ ClCH2 CH2 Cl/ DTAB

Water/ ClCH2 CH2 Cl/ DTAB

Solvent

25–50◦ C

60◦ C

65 C

◦

C

C

C

C

P(Cy)3

Ru

PPh3 P(Cy)3

Ru

PPh3

P(Cy)3 Ph Ru H C P(Cy)3 C

P(Cy)3 Ph Ru H Cl P(Cy)3

Cl

Temperature Initiator

Ph Ph

Ph Ph

166

165

164

Reference

87

11

12

13

Mannose

Galactose

Galactose

O O

O

O

O

O

OO

O

OH

O

O

O

OH

O

O N

H

H

O O

O

O

OH OH

O

O O

O

O

O

O

Toluene

Toluene

MeOH/ H2 O/ (CH2 Cl2 )

Room temperature

Room temperature

Room temperature

O

iPr

O

iPr

O

iPr N Ph Me Me Mo

O

iPr N Ph Me Me Mo

P(Cy)3 Ph Ru H Cl P(Cy)3

Cl

(continued)

168

168

167

88 14

15

Ribonic acid

Entry

Mannose

Carbohydrate

O

O

O

O

O

O

O

O

O

Monomer

O

O

O

TABLE 1.8 Glycomonomers Polymerized via ROMP (Continued)

Toluene

Toluene

Solvent

Room temperature

Room temperature

O

iPr

O

iPr

Temperature Initiator

O

iPr N Ph Me Me Mo

O

iPr N Ph Me Me Mo

168

168

Reference

89

RING-OPENING METATHESIS POLYMERIZATION (ROMP)

O H O

O

H O

O O H C R

RuCl

H O

O H H OO

H H OO

O O H C R

O

O O R

CH

O H H OO

O O H C R

O O R H C

H O

R=

O OH

OH OH OH

FIGURE 1.25 ROMP of an asymmetric glycomonomer for the synthesis of stereochemically diverse glycopolymers [163].

An interesting feature of ROMP-synthesized polymers for biological applications is the simultaneous existence of cis- and trans-isomers in the backbone, which is supposed to enhance the interaction with lectins. To test the hypothesis that variety in polymer geometries enhances the interaction with lectins, the isomer diversity was further increased by employing asymmetric glycomonomers (Table 1.8, entries 5 and 6). Indeed, stereochemically diverse glycopolymers based on 5 and 6 showed an increased activity in their binding to Con A (Fig. 1.25) [163]. To date, most glycomonomers based on 7-oxobornene have been polymerized in water with RuCl3 as initiator. The molecular weights obtained were typically around 106 g mol−1 . The polymerizations, however, were not living. To address the need to generate glycopolymers of different molecular weights via ROMP the Grubbs ruthenium alkylidene catalyst was employed; however, a challenge was presented by the contrasting solubilities of catalyst and monomer. Finally, emulsion conditions were employed with dodecyltrimethylammonium bromide (DTAB) in a 1,2-dichloroethane/water mixture. These conditions were employed using sulfated glycomonomers with 7-oxobornene (Table 1.8, entry 7) as the polymerizing moiety, but also norbonene (Table 1.8, entry 8), and the resulting polymers were found to be effective P-selectin inhibitors with the disulfated compounds showing higher activity [164]. The inhibition of L-selectin was achieved by the norbonene derivate with a disulfated trisaccharide side group (Table 1.8, entry 9). The polymerization was carried out at a low monomer to Grubbs initiator ratio to obtain oligomeric species. The narrow molecular weight distribution of PDI < 1.2 indicated a well-controlled process [165]. The control of molecular weight in conjunction with narrow molecular weight distributions is an attractive feature of living techniques such as ROMP. A detailed study into the livingness of the ruthenium carbene catalyzed polymerization of glycomonomers was first carried out by Fraser and Grubbs. A set of protected and unprotected norbornene derivatives (Table 1.8, entry 10) was polymerized using an active and a less active ruthenium carbene catalyst (Fig. 1.26) [166]. The less active catalyst A (Fig. 1.26) was only able to initiate the polymerization of the acetylated monomer II, albeit forming insoluble gel under certain circumstances. Better results were obtained using the more reactive catalyst B, but the outcome varied depending on the solvent. While heating was usually not required with catalyst B, lower PDIs (<1.2) were observed at elevated temperatures. The monomers II–V (Fig. 1.26) could all be successfully polymerized at 50◦ C to give narrow molecular weight distributions, but the reaction rates were strikingly different with polymerization times ranging from 5 min to several days. A related catalyst was utilized

90

SYNTHESIS OF GLYCOPOLYMERS

PPh3 Cl RO HN O

Ru

Cl

O

PPh OR OR OR'

R=R'=H R=R'=Ac R=R'=CH Ph R=R'=SiEt R=H; R'=CPh

Ph

Ru

n

A

or P(Cy) Cl

I II III IV V

Ph Ph

RO HN

Ru

Ph Ph

P(Cy)

B

Cl

O

Ph

O OR OR OR'

FIGURE 1.26 ROMP of norbornene derivatives with either ruthenium carbene catalyst A or B (A less active than B) by Fraser and Grubbs [166].

to synthesize glycopolymers with a range of molecular weights. The targeted number of repeating units was obtained by adjusting the monomer-to-catalyst ratio. The mannose-containing polymer (Table 1.8, entry 11) was tested regarding its activity to inhibit erythrocyte agglutination. The length of the molecular weight was indeed found to influence the relative potency. Strong effects compared to the monomeric units were found at low molecular weights, while no improvement was obtained when the number of repeating units exceeded 50 [167]. A molybdenum catalyst was shown to convey living characteristics to the polymerization of a selection of norbonene-based glycomonomers (Table 1.8, entries 12–13). The livingness was not only demonstrated by the ability to target a particular molecular weight by adjusting the monomer–catalyst ratio, but also by the capability to synthesize multiblock copolymers by merely adding more monomer. An interesting observation was the influence of the cleavage procedure to remove the metal from the polymer in a Wittig-type reaction with aldehydes. While some reactions caused the broadening of the molecular weight distribution, probably due to crosslinking, other procedures maintained PDIs below 1.1 [168]. 1.8 POSTFUNCTIONALIZATION OF PREFORMED POLYMERS USING SUGAR MOIETIES The modification of preformed polymers using saccharide-containing regents offers an excellent alternative synthetic route to obtaining glycopolymers. The postfunctionalization approach is convenient for producing libraries of glycopolymers with the same macromolecular features by attaching different sugar moieties to preformed polymer scaffolds. Problems associated with the synthesis and purification of glycomonomers, which are often prone to self-polymerization, are also circumvented. Each postfunctionalization reaction explored in this section is summarized in Table 1.9 with details on the structure of the sugar moeity attached, the relevant reaction conditions, and the corresponding reference. The reactions appear in the same order as in the text. Unless otherwise specified, “R” in the figures of this section refers to the saccharide unit.

91

Condensation DMSO

Condensation DMSO

(GlcNAc)3

Solventa

Iso-LacNAc

Reaction Condensation DMSO

Sugar Moiety to be Attached

Synthesis of Glycopolymers via Postmodification

LacNAc

Carbohydrate

TABLE 1.9

RT

RT

RT

Temperature

BOP, HOBt

BOP, HOBt

BOP, HOBt

Other Reactantsb

(continued)

29

29

29

Reference

92 Acryloyl chloride + amine

Galactosamine

Carbonate buffer

0–5◦ C

TEA and ethyl chloroformate

−8◦ C to RT

Acid + amine

Glucosamine

DMF

BOP, HOBt

BOP, HOBt

Other Reactantsb

RT

RT

Temperature

Condensation DMSO

Solventa

GlcNAc

Reaction Condensation DMSO

Sugar Moiety to be Attached

Synthesis of Glycopolymers via Postmodification (Continued)

(GlcNAc)2

Carbohydrate

TABLE 1.9

171

169, 170

29

29

Reference

93

DMF

DMSO

NHS + amine

NHS + amine

Anhydride + amine

Mannose

GalNAc

Lactose

Anhydrous DMSO

Carbonate buffer

Acryloyl chloride + amine

Glucosamine

RT, under N2

RT

RT

0–5◦ C

NMM

(continued)

175

173

172

171

94 Nitrophenyl carbonate + amine

Nitrophenyl carbonate + amine

Nitrophenyl carbonate + amine

Glucose

Galactose

Glucose

Reaction Nitrophenyl carbonate + amine

Sugar Moiety to be Attached

Synthesis of Glycopolymers via Postmodification (Continued)

Glucosamine

Carbohydrate

TABLE 1.9

DMSO

DMSO

DMSO

DMSO

Solventa

177

177

177

70◦ C

70◦ C

70◦ C

Reference 176

Other Reactantsb

70◦ C

Temperature

95

Azide–alkyne DMSO click

Azide–alkyne DMSO click

Azide–alkyne DMSO click

Azide–alkyne DMSO click

Azide–alkyne DMSO click

Mannose

Galactose

Mannose

Galactose

Galactose

(PPh3 )3 CuBr, TEA

(PPh3 )3 CuBr, TEA or CuBr, TBTA, TEA or CuBr, bipy, TEA

RT or 60◦ C

(PPh3 )3 CuBr, TEA

(PPh3 )3 CuBr, TEA

(PPh3 )3 CuBr, TEA

RT

RT

RT

RT

(continued)

178d, 178e

178c

178b–d

178a

178a

96 Thiol-ene click

Thiol-ene

Glucose

Glucose

THF

DMF

Azide–alkyne DMSO click

Solventa

Lactose

Reaction Azide–alkyne DMSO click

Sugar Moiety to be Attached

Synthesis of Glycopolymers via Postmodification (Continued)

Mannose

Carbohydrate

TABLE 1.9

UV, under argon atmosphere

DMPA

(PPh3 )3 CuBr, TEA or CuBr, TBTA, TEA or CuBr, bipy, TEA

RT or 60◦ C

RT, UV

(PPh3 )3 CuBr, TEA or CuBr, TBTA, TEA or CuBr, bipy, TEA

Other Reactantsb

RT or 60 C

◦

Temperature

180, 181

179

178d, 178e

178d, 178e

Reference

97

DMF or THF

Thiol + bromide

Glucose

RT

80◦ C

K2 CO3

CSA or TfOH

183

182

b BOP

a DMSO

= dimethyl sulfoxide; DMF = N,N-dimethyl formamide; THF = tetrahydrofuran; DMAc = N,N-dimethyl acetamide. = benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate; HOBt = 1-hydroxy-benzotriazole; TEA = triethylamine; NMM = Nmethylmorpholine; TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; DMPA = 2,2-dimethoxy-2-phenyl-acetophenone; CSA = (1S)-(+)-10-camphorsulfonic acid; TfOH = trifluoromethanesulfonic acid.

DMAc

Sugar oxazoline + OH

GlcNAc

98

SYNTHESIS OF GLYCOPOLYMERS

O

O

H N n + R

O

OH

H N

x

Condensation

NH

O

H N

O

N H

y

R

O

OH

FIGURE 1.27 Synthesis of glycopolymers carrying N-acetyllactosamine and related compounds [29].

1.8.1 Amide Linkage The earliest attempts to modify preformed polymers with sugar moieties focused on aminosaccharides, which were attached to the polymer backbone via an amide linkage. Polymers with pendant carboxylic acid, N-hydroxysuccinimide (NHS) and other functionalities are suitable candidates for reaction with aminosaccharides. Kobayashi and co-workers [29] reported the synthesis of glycopolymers carrying N-acetyllactosamine and other related compounds. As shown in Figure 1.27, condensation reactions between glycosylamines and the carboxyl groups of poly(lglutamic acid) were used. The glycosylamines were synthesized by reacting sugars with ammonium hydrogen carbonate. The condensation reaction between carboxyl group and amino functionality was carried out in the presence of benzotriazole-1-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole (HOBt). In this report, LacNAc-NH2 , iso-LacNAc-NH2 , GlcNAc-NH2 , (GlcNAc)2 -NH2 , and (GlcNAc)3 -NH2 were all used to make the targeted glycopolymers with the degree of substitution ranging from 31 to 78%. Copolymers of N-vinylpyrrolidone (NVP) and tert-butyl methacrylate (t-BMA) were synthesized by Mart´ınez and co-workers. They were hydrolysized under acidic conditions to achieve terpolymers with pendent carboxylic acid groups, and then modified with 2-amino-2-deoxy-d-glucose to afford new sugar-carrying amphiphiles. The coupling reaction was carried out in DMF catalyzed with triethylamine and ethyl chloroformate at −8◦ C for 2 h and then at room temperature for 18 h. Finally about 50% of the carboxylic acid groups were modified with glucosamine (Fig. 1.28) [169]. S´achez-Chaves and co-workers synthesized poly(vinyl alcohol) (PVA) partially functionalized with monosuccinate groups by reaction of PVA with succinic

y

x O

N

O

O

x O

N

y-n O

OO

x

n OH

O

N

y-n O

OO

n -a

a

OH O

NH R

FIGURE 1.28 Synthesis of glycopolymers from acid functional polymers [169].

99

POSTFUNCTIONALIZATION OF PREFORMED POLYMERS USING SUGAR MOIETIES

x O

y

x-y

Cl

O

OH

O

NH R

FIGURE 1.29 Synthesis of macromonomers with glucose and galactose end-functionality [171].

anhydride using triethylamine as catalyst and N-methyl-2-pyrrolidone as solvent. The polymers were then modified with 2-amino-2-deoxy-d-glucose to afford the desired glycopolymers [170]. In another report, hydrophilic polyacrylamide compounds possessing glucose (PAAm–glucose) and galactose (PAAm–galactose) as pendent groups were synthesized from poly(acryloyl chloride) by Kodama and co-workers (Fig. 1.29). Poly(acryloyl chloride) was first polymerized in dry 1,4-dioxane via free radical polymerization. Glucosamine and galactosamine hydrochloride in an alkaline medium such as bicarbonate buffer, were then reacted with the poly(acryloyl chloride) to form polyacrylamide displaying glucose (PAAm–glucose) and galactose (PAAm–galactose) residues. The degree of glucose and galactose substitution was 53.2 and 41.6%, respectively [171]. Strong and co-workers [172] synthesized a N-hydroxysuccinimide (NHS)substituted materials via ring-opening metathesis polymerization, which were then treated with a mannose derivative containing an aglycon linker terminating with a primary amine. The reaction was carried out at room temperature under nitrogen atmosphere for 24 h (Fig. 1.30). Baek and Roy also utilized the coupling reaction between NHS and amine groups by first synthesizing poly(N-acryloxysuccinimide) via free radical polymerization, and conjugating the polymer with the aminated carbohydrate ligand 3-(2aminoethylthio)propyl ␤-d-Gal-(1-3)-␣-d-GalNAc. The reaction was performed in DMSO at room temperature followed by quenching with NH4 OH (Fig. 1.31). Furthermore, random glycopolymers bearing T-antigen were also synthesized in a similar manner [173]. Poly(N-acryloxysuccinimide) (polyNAS) with narrow molecular weight distributions were also successfully prepared by atom transfer radical polymerization (ATRP).

OH

Ph

n O O O

+ O

N

HO HO

Ph HO O

n O

OH NH2 O

HO HO

HO O HN O

FIGURE 1.30 Synthesis of macromonomers with mannose end-functionality [172].

100

SYNTHESIS OF GLYCOPOLYMERS

O

O O

O

N

OH OH

OH OH

y

x

NH

O

O

NH +

OH

O

AcHN

OH

AcHN

S

O

FIGURE 1.31 Graft conjugation oxysuccinimide) [173].

O

O

R1 R 2

O

O

of aminated

sugar

moiety

onto

O n

N

poly(N-acryl

R1 = R 2 = ONa or O

NH2

R1 = ONa and R 2 = N

NH

O O

O

HO

S

O

HN

O

O

y

x

OH OH

OH OH

O

HO

O n

OH

OH

HO

O

(or vice versa)

OH

O HO

NH OH

OH OH

O

FIGURE 1.32 Synthesis of galactosylated N-vinylpyrrolidone-maleic acid copolymers [175].

Well-defined glycoconjugate polyacrylamides were then generated by substituting N-oxysuccimide units with galactosamine followed by reaction with ethanolamine [174]. Auzely-Velty et al. prepared a copolymer of N-vinylpyrrolidone and maleicanhydride via radical polymerization (Fig. 1.32). The polymer was dried over P2 O5 in anhydrous DMSO to avoid the hydrolysis of anhydrides, and then reacted with N-(4-aminobutyl)-O-␤-d-galactopyranosyl-(1-4)-d-gluconamide overnight under nitrogen. The solvent was evaporated and diluted with water and reacted with NaOH solution for another 10 h. The reaction was based on the nucleophilic attack of the amino end group on the anhydride functions. Only one of the two carboxylic groups per maleic monomer was modified, and glycopolymers with a substitution degree ranging from 3 to 50% were obtained. Despite the relatively low substitution ratio, the resulting copolymer with pendant galactose moieties showed good inhibitory properties against model RCA120 lectin [175]. Polymers with pendant hydroxyl groups have also been used as a backbone to attach saccharides. Garc´ıa-Oteiza et al. [176] reported that PVA samples partially functionalized with 4-nitrophenyl carbonate groups could be obtained by reaction of PVA with 4-nitrophenyl chloroformate using pyridine as a catalyst. Glucosaminecarrying PVA was then generated by reacting the above polymer with 2-amino-2deoxy-d-glucose in DMSO at 70◦ C (Fig. 1.33).

O H

C

OH +

Cl

C

O O

NO2

H

C

O

C

R O

NO2

NH2

O H

C

O

C

H N

R

FIGURE 1.33 Synthesis of glycopolymers from 4-nitrophenyl carbonate functionalized polymer [176].

101

POSTFUNCTIONALIZATION OF PREFORMED POLYMERS USING SUGAR MOIETIES

n O

+ R

O O

NH

O

n H N

R

O

NO

FIGURE 1.34 Synthesis of glycopolymers from p-nitrophenyl chloroformate functionalized polymer [177].

Similarly, Cerrada et al. reported another strategy for synthesizing glycopolymers based on vinyl alcohol polymers (Fig. 1.34) [177]. Commercially available random ethylene-vinyl alcohol copolymer, EVOH, was used and modified with p-nitrophenyl chloroformate. Subsequently, the glycosylation was carried out in DMSO with different aminosaccharides at 70◦ C for 24 h. The degree of modification ranged from 41 to 61%. The glycopolymer showed specific interaction with certain lectins. 1.8.2 Click Approach Although the aforementioned reactions that attach saccharide units to polymers via amide linkages have been used with significant success, many of them suffer from long reaction times, low conversions, and high sensitivity to moisture and oxygen. Consequently, researchers have sought faster, more efficient reactions with simpler workups for generating well-defined glycopolymers via a postfunctionalization approach. Haddleton and co-workers presented a series of reports on the construction of glycopolymers from alkyne backbone-functional polymers via Cu-catalyzed azide–alkyne click (CuAAC) chemistry (Fig. 1.35) [178]. Well-defined polymer backbones with alkyne functionalities were first synthesized via living radical polymerization or catalytic chain transfer polymerization. Different sugar azides (mannose, galactose, lactose) were made and “clicked” to the backbone. In this fashion, a variety of well-defined glycopolymers were obtained and tested for their binding abilities with specific lectins. Detailed information on each of these reactions is provided in Table 1.9.

n O

n

+ R-N3

O

O R

O

N N N

FIGURE 1.35 Synthesis of glycopolymers via Cu-catalyzed azide–alkyne click chemistry [178].

102

SYNTHESIS OF GLYCOPOLYMERS

OH +

n O

O

HO HO

O

OH O

n SH

O

O

O

O

O

OH O

HO HO

OH

S

FIGURE 1.36 Synthesis of glycopolymers via thiol-ene click reaction [179].

Another click reaction called thiol–ene coupling or thiol–ene click has been recently employed by researchers for the synthesis of glycopolymers (Fig. 1.36). Thiol–ene coupling is highly efficient and tolerant to a wide range of functional groups and is compatible with water and oxygen. In addition, the coupling reaction is simple and biologically friendly (metal free). Commercially available poly(hydroxyethyl methacylate) and block copolymers of poly(ethylene glycol)methacrylate and HEMA synthesized via RAFT polymerization were used by Stenzel and co-workers as a backbone for the synthesis of glycopolymers. The hydroxyl groups were converted to alkene functionalities via reaction with 4-pentenoic anhydride. Homopolymers and block copolymers bearing carbohydrate side groups were then obtained by grafting glucothiose onto the alkene-functionalized scaffolds via a thiol–ene click reaction in the presence of a photoinitiator in DMF for 2 h. Complete conversions were observed in both cases. The resulting copolymer was then used to form thermoresponsive micelles as a potential drug carrier [179]. In a report by You and Schlaad, a thioglucose derivative was effectively grafted onto the double bonds of a poly(1,2-butadiene)-b-poly(styrene) through photoaddition at room temperature with AIBN in dry THF (Fig. 1.37) [180]. The same researchers also demonstrated the synthesis of a series of poly(2-(isopropyl/3-butenyl)2-oxazolin)s by cationic ring-opening polymerization of a monomer mixture of 2-isopropyl-2-oxazoline (IPOX) and 2-(3-butenyl)-2-oxazoline (BOX). The copolymer was then modified by photoaddition of a thioglucose derivate, 2,3,4,6-tetra-Oacetyl-1-thio-␤-d-glucopyranose, onto the vinyl double bonds in THF for 24 h. The acetyl protecting groups were quantitatively removed by alkaline treatment with

N O

N x O

N

y O

N x O

y

S R

FIGURE 1.37 Synthesis of glucose-containing polymers [180].

103

POSTFUNCTIONALIZATION OF PREFORMED POLYMERS USING SUGAR MOIETIES

OAc x OH

AcO AcO

x-y O

OH

y OH

HO HO

O

x-y O

y OH

O NHAc

NHAc

FIGURE 1.38 Synthesis of GlcNAc-substituted PVA [182].

sodium methoxide at room temperature to afford the desired glucose-containing polymer [181]. 1.8.3 Other Nonclick Approaches Takasu and co-workers established a procedure for chemical modification of poly(vinyl alcohol) (PVA) by a glycosidation reaction of hydroxyl groups in PVA with triacetylated sugar oxazoline. In this way, triacetylated N-acetyl-d-glucosamine (GlcNAc) was introduced onto a PVA backbone selectively via a ␤-O-glycoside linkage. Deacetylation of triacetylated GlcNAc-substituted PVA resulted in GlcNAcsubstituted PVA with a degree of substitution ranging from 0.07 to 0.24 (Fig. 1.38) [182]. Xue and co-workers have reported that poly(fluorenes) prepared by Sonogashira coupling of bromo-alkane functionalized monomers can be glycosylated simply by the reaction of the bromo-groups with a thiosugar. Well-defined glucosecarrying polymers were prepared by coupling poly[(9,9-bis(6 -bromohexyl)-2,7fluorenylene)-alt-1,4-phenylene] with an excess of 1-thio-␤-d-glucose tetraacetate under basic conditions. Polymers were then deacetylated in methanol and methylene chloride containing sodium methoxide, affording the fluorene-based conjugated glycopolymers. The glycopolymer was also obtained by the direct reaction of poly[(9,9-bis(6 -bromohexyl)-2,7-fluorenylene)-alt-1,4-phenylene] with 1-thio-␤-dglucose sodium salt hydrate under basic conditions (Fig. 1.39) [183]. There are some other methods to prepare glycopolymers, for example, by using carbohydrates bearing aldehyde groups that react efficiently with polymers bearing pendant amino residues [184]. Poly(ethylene terephthalate) (PET) fibers can be

Br

Br

S O

S OH

OH

O

HO HO OH

HO

OH OH

FIGURE 1.39 Synthesis of fluorene-based polymers bearing glucose pendants [183].

104

SYNTHESIS OF GLYCOPOLYMERS

substituted photochemically with glycosyl azides [185], although the degree of functionalization is not quantitative.

1.9 CONCLUSIONS This chapter summarizes the cornu copiae of glycopolymer structures that have been synthesized via a wide array of polymerization and postpolymerization strategies. Linear polymers with controlled molecular weights and low-molecular-weight distributions are readily available by applying many of these techniques; however, the synthesis of glycopolymers is currently dominated by living radical procedures, with most publications detailing glycopolymers produced via ATRP or RAFT polymerization. The robustness of these techniques and their provision for molecular weight control by simply adjusting the conversion and the concentration of controlling agent undoubtedly contribute to their popularity. The ability to generate complex architectures is also offered by many of the techniques discussed in this chapter; however, relatively few publications deal with polymer structures beyond simple block copolymers. Reports on star polymers and comb polymers are limited, and there is still room to explore more complex polymer architectures. It would also appear that great possibilities remain unexplored in the area of bioconjugation and other biomedical applications of glycopolymers with well-defined architectures. A vast array of natural saccharide-containing molecules are ubiquitous in living systems, and present exciting possibilities as in vivo targets for glycopolymer-containing bioconjugates and as model species that may be copied by polymer chemists in the development of new biomimetics. It is reasonable to propose that the continued evolution of living/controlled polymerization strategies will make such structures attainable. Despite the breadth of polymers that are accessible via the polymerization of sugar-containing species, there are still limitations associated with each of the polymerization strategies. Significant developments in the area of postfunctionalization present great opportunities for extending the field of glycopolymer synthesis beyond what would be possible using existing polymerization strategies. Postfunctionalizations with carbohydrates can be highly effective where solubility issues and chain transfer events prevent the synthesis of glycopolymers directly from glycomonomers. In summary, polymer science now offers a substantial toolbox of synthetic options to generate glycopolymers with defined molecular weights and polymer architectures for a range of applications, and there exists great potential for further developments in this area.

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106

SYNTHESIS OF GLYCOPOLYMERS

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CHAPTER 2

BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES QIAN YANG Lehrstuhl fur ¨ Technische Chemie II, Universitat ¨ Duisburg-Essen, Essen, Germany

2.1 Introduction 2.2 Synthesis of Block Glyco-Copolymers 2.2.1 Conventional Free Radical Polymerization 2.2.2 Atom Transfer Radical Polymerization (ATRP) 2.2.3 Reversible Addition–Fragmentation Chain Transfer Process (RAFT) 2.2.4 Other Methods 2.3 pH-Sensitive Glycopolymers 2.4 Temperature-Sensitive Glycopolymers 2.5 Conclusions and Future Trends Acknowledgment References

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2.1 INTRODUCTION Carbohydrates, ubiquitous in living things, have been found on the surface of nearly every cell in the form of polysaccharides, glycoproteins, glycolipids, or/and other glycoconjugates [1, 2]. The carbohydrates on the external cell membrane, known as the glycocalyx, play essential roles in many biological functions that can be classified into two aspects. First, they can serve as sites for the docking of other cells, biomolecules, and pathogens in a more or less specific recognition process [3]. Second, they can contribute to steric repulsion that prevents undesirable nonspecific adhesion of other proteins and cells [4]. Both of these aspects rely on the Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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carbohydrate–protein interactions in which the mechanisms remain controversial even today. Though the mechanisms and the structures of carbohydrates are still not well known, these carbohydrate–protein interactions are widely accepted to be the key in a variety of biological processes and the first step in numerous phenomena based on cell–cell interactions, such as blood coagulation, immune response, viral infection, inflammation, embryogenesis, and intercellular signal transfer [5–7]. Recently, there has been great interest in the carbohydrate–protein interactions and many efforts have been made to reveal the underlying essence. Although the mechanism is not well known, it is clear that, in nature, carbohydrates binding species typically aggregate into higher order oligomeric structures both for prevention of undesirable nonspecific adhesion and for specific recognition [4, 8]. Actually, highly aggregated carbohydrate ligands, just like those glycocalyx on the cell membrane surfaces, result in not only the enhancement of binding strength in specific recognition against proteins but also the minimization of the nonspecific protein adsorption. The binding strength and also its specificity are remarkably improved by multivalent interactions, which are found quite regularly in biosystems [9]. The mechanism by which multivalent ligands act is still not very well known, but it has been accepted that this “cluster glycoside effect” dominates the carbohydrate–protein interaction [10]. Consequently, a large number of different synthetic multivalent glycoligands (such as glycoclusters, glycodendrimers, and glycopolymers) have been designed to interfere effectively with the carbohydrate–protein interactions and to facilitate the investigation of the multiple interactions occurring during these molecular recognition events [2, 11–19]. Among these manmade multivalent carbohydrate ligands, glycopolymers continue to be attractive because of very high valency, easy to control molecular structure, and facile to vary the species of the sugar ligands displayer on the polymer backbone. In the last decade, a great number of sugar-containing monomers and their polymers are expected to display complex functionalities, similar to those of natural glycoconjugates, which might be able to mimic, or even exceed, their performance in specific applications [20]. Synthetic carbohydrate-based copolymers having pendant sugar residues are of great interest, not only as simplified models of mimicking biopolymers bearing oligosaccharides but also as artificial glycoconjugates in biochemistry and medicine. Moreover, synthetic carbohydrate containing copolymers that are biocompatible and biodegradable are increasingly found applications in many fields. They could serve as enzyme/virus/toxin inhibitor. Sialyllactose-bearing glycopolymers are known as an effective inhibitor for the infection of influenza virus. The activity of the glycopolymer can be 103 times higher than that of the oligosaccharide itself (see in Fig. 2.1) [21]. As shown in Figure 2.2, ␤-d-galactosamine-functionalized glycopolymers with poly(l-glutamic acid) backbone showed significant inhibiting effect to cholera toxin, and this inhibition relies on competitive mechanism rather than steric stabilization [22]. The specific interactions between sugars and proteins also give a chance to glycopolymer for applications in tissue engineering, affinity matrix, and drug/gene delivery devices. Glyco-copolymers carrying galactose as the pendant group are known to have specific interaction with the asialoglycoprotein receptor on hepatocytes and hepatocarcinomas [23]. Copolymers containing galactose and mannosamine have also been synthesized and interfered in affinity matrices for culture of hepatocytes and targeted to human glioblastoma cells and macrophages, respectively

INTRODUCTION

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FIGURE 2.1 Poly(p-vinylbenzoyl-sialyllactosylamine) with ␣-(2-3) and ␤-(2-6) linked sialyllactose.

[24–28]. Carbohydrate residues on glycopolymers can selectively bind to pathogens, which makes it possible to employ glycopolymer for probing nosogenesis. Fluorescent polymers with carbohydrates side groups can be a versatile detection method for bacteria. The mannose-modified poly(p-phenylene ethynylene) (PPE) was used for the detection of Escherichia coli by multivalent interactions (Fig. 2.3). This detection

FIGURE 2.2 Poly(l-glutamic acid) with ␤-d-galactosamine side groups is effective inhibitor of cholera toxin.

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FIGURE 2.3 Poly(p-phenylene ethynylene) (PPE) with pendant mannose groups (left) and the aggregation of E. coli by mannosylated PPE (right).

is highly selective and with galactose on the polymer backbone there was no bacteria stained with fluorescence [29]. 2.2 SYNTHESIS OF BLOCK GLYCO-COPOLYMERS Various polymerization techniques have been applied to synthesize block glycocopolymers, including conventional radical polymerization, atom transfer radical polymerization (ATRP), reversible addition–fragmentation chain transfer (RAFT) process, nitroxide-mediated polymerization, and ring-opening polymerization. Other methods such as polymer analogous reaction and click chemistry are also employed for glyco-copolymer construction. 2.2.1 Conventional Free Radical Polymerization Conventional free radical polymerization is the most convenient way for synthesis of glyco-copolymers. It has many advantages over other mechanisms such as being easy to carry out, polymerization under mild reaction conditions, and tolerance to protonic impurities. It can also be used for a wide range of monomer types and, normally, the sugar-containing monomers do not need protection. However, conventional free radical polymerization has little control over polymer chain length and structure. For glyco-copolymer fabrication, random copolymers always obtained by conventional free radical polymerization and the content of glycopolymer units can be hardly controlled. David et al. copolymerized galactosamine, lactose, and triantennary galactose containing monomers with N-(2-hydroxypropyl)methacrylamide (HPMA) and 5-[3-(methacryloylaminopropyl)-thioureidyl] fluorescein (MA–AP–FITC) (see Fig. 2.4) [30]. The recognition of these three triblock copolymers to human hepatocarcinoma HepG2 cells was investigated. The results show that the trivalent galactose and lactose-containing copolymers bind preferentially to HepG2. The cells endocytose the bound polymers and fluorescence can be seen inside the cells. These glyco-copolymers may be useful as a drug delivery system with specific binding affinity to hepatocytes.

SYNTHESIS OF BLOCK GLYCO-COPOLYMERS

123

FIGURE 2.4 Structure of saccharide-containing HPMA copolymers and the corresponding amino-sugar.

2.2.2 Atom Transfer Radical Polymerization (ATRP) In recent years ATRP has been widely employed for the construction of welldefined copolymers. Compared to conventional free radical polymerization, the living/controlled character of the ATRP achieves polymers with a low polydispersity (M W /M n ) with end-functionalization and so can be used as macroinitiators for the formation of di- and triblock copolymers [31–33]. Moreover, ATRP is much more tolerant to monomers with variety of functional groups than other living systems such as living anionic and cationic polymerizations. Dai and co-workers synthesized a biodegradable star-shape block glycocopolymer by ATRP [34]. As shown in Figure 2.5, first ring-opening polymerization

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BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

FIGURE 2.5 Synthesis of star-shaped SPCL–PGAMA copolymers.

125

SYNTHESIS OF BLOCK GLYCO-COPOLYMERS

of ε-caprolactone was carried out to synthesize poly(ε-caprolactone)-bpoly(gluconamidoethyl methacrylate) (SPCL–PGAMA), a new class of biodegradable and biomimetic star-shaped SPCL–PGAMA block copolymers via sequential process followed by direct ATRP (and/or metal-catalyzed living radical polymerization, ATRP) of unprotected GAMA glycomonomer. Narain and Armes synthesized methacrylate-based glyco-copolymers bearing glucose and lactose side groups by ATRP [35]. The glycomonomers in this study were not protected and were used directly in ATRP, thus introducing a facile procedure for glycopolymer synthesis. The di- or triblock copolymers with poly(ethylene oxide) [PEO], poly(propylene oxide) [PPO], 2-(diethylamino)ethyl methacrylate [DEA], glycerol monomethacrylate [GMA], 2-(diisopropylaminoethyl methacrylate [DPA], and poly-(epsilon-caprolactone) [PCL] showed well-tailored structure and the polydispersity indexes were lower than 1.4 (see Table 2.1). TABLE 2.1 Molecular Weight of GAMA- and 2-lactobionamidoethyl methacrylate (LAMA) based Diblock and Triblock Copolymers Block Copolymer Composition

ATRP Solvent Composition

Yielda (%)

PEO23 –GAMA50 PEO113 –GAMA50 PEO23 –GAMA30 –DEA60 PEO23 –GAMA30 –DEA80 PEO23 –GAMA50 –DEA100 PEO23 –GAMA50 –GMA60 PEO23 –GAMA30 –GMA60 PEO23 –GAMA30 –DPA80 PEO23 –GAMA30 –DPA60 PPO33 –GAMA50 PPO33 –GAMA30 PPO33 –GAMA20 GAMA15 –PCL18 –GAMA15 GAMA50 –PCL18 –GAMA50

Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol 4:1 IPA/H2 O 4:1 IPA/H2 O

75 76 75 73 80 79 81 75 74 75 79 74 82 78

Copolymer Type

Solvent Composition

PEO23 –LAMA30 –DEA50 Ald–LAMA25 –DEA50 PEO23 –LAMA30 –GAMA30 Ald–LAMA25 –GMA60

3:2 methanol/water 3:2 methanol/water NMP NMP

Mn b

Mw /Mn

11,400 16,000 27,400 17,200 37,600 25,200 19,500

1.23 1.19 1.29 1.28 1.37 1.29 1.32

23,000 15,500 12,850 14,500 24,600

1.11 1.22 1.23 1.15 1.12

Solubility in Waterc Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes

Mn of Final Mw /Mn of Mn of LAMA Block Final Block Homopolymer Copolymer Copolymer 11,400 10,600 10,100

17,900 17,300 21,200 18,100

1.34 1.30 1.28 1.29

a Final yield obtained after purification using basic alumina to remove the spent ATRP catalyst. The actual

NMR conversions were close to 95%. b Determined by GPC using DMF containing 0.01 M LiBr at 70◦ C and calibrated with poly(methyl methacrylate) standards except entries 1 and 2, which were analyzed by aqueous GPC [0.20 M sodium nitrate and 0.01 M sodium dihydrogen phosphate as eluent at pH 7 with poly(ethylene oxide) calibration standards]. c At pH 7 and 20◦ C. Source: Reproduced with permission from [35].

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FIGURE 2.6 Synthesis of copolymer from mannose-containing monomer and fluorescent rhodamine B comonomer by ATRP.

Geng and co-workers synthesized a well-defined maleimide-terminated glycocopolymer by ATRP [36]. As shown in Figure 2.6, mannose-containing monomer (2) was polymerized with fluorescent rhodamine B comonomer (9) initiating by the maleimide-protected initiator 8. After deprotection by retro Diels–Alder reactions, the glyco-copolymer was attached to bovine serum albumin (BSA) to obtain a glycoprotein mimics. The glyco-copolymer showed a linear increase of molecular weight with polymerization time. The molecular weight can be controlled between 8 and 30 kDa, and relatively low polydispersities, which were lower than 1.3 (1.20–1.28), was achieved (Fig. 2.7). Zhou et al. designed and synthesized a special star-shaped biodegradable and biomimetic poly(ε-caprolactone)-block-poly(lactobionamidoethylmethacrylate) (SPCL–PLAMA) block copolymers by sequential ring-opening polymerization of εcaprolactone and by the subsequent direct ATRP of unprotected lactobionamidoethyl methacrylate (LAMA) glycomonomer [37]. As shown in Figure 2.8, a star-shaped PCL polymer that was terminated with four hydroxyl end groups (SPCL-OH) was synthesized by ring-opening polymerization of ε-caprolactone [38–40]. Then the ATRP initiator was immobilized to the end of this SPCL–OH polymer by the reaction between the hydroxyl groups and 2-bromo-2-methylpropionate (SPCL-Br). Afterward, ATRP of LAMA was carried out from the macroinitiator SPCL-Br. The molecular weight of the SPCL–PLAMA can be controlled linearly by the molar ratio between LAMA glycomonomer and SPCL-Br macroinitiator as shown in Table 2.2. Moreover, this glyco-copolymer exhibits self-assembly in aqueous solution, and the aggregates shape changed from spherical micelles to vesicles with the increase of weight fractions of hydrophobic PCL segment in the SPCL–PLAMA copolymer (Fig. 2.9).

SYNTHESIS OF BLOCK GLYCO-COPOLYMERS

127

FIGURE 2.7 ATRP of mannose-containing monomer and fluorescent rhodamine B initiated by the maleimide-protected initiator.

2.2.3 Reversible Addition–Fragmentation Chain Transfer Process (RAFT) The RAFT process has been also considered as a very promising living polymerization technique for glyco-copolymers because of its tolerance to reaction conditions and glycomonomers. On the other hand, RAFT offers excellent control in aqueous media, which is a good solvent for many glycomonomers. Moreover, necessarily needed heavy-metal catalysts in the ATRP system do not appear in RAFT, which makes it suitable for many biomedical applications. Deng and co-workers synthesized homopolymers from 3-gluconamidopropyl methacrylamide (GAPMA) and 2-gluconamidoethyl methacrylamide (GAEMA) by using 4-cyanopentanoic acid dithiobenzoate as the chain transfer agent (CTA) [41]. After that, the glycopolymers were used as macro-CTA and copolymerized with methacryloyloxyethyl phosphorylcholine (MPC). As shown in Figure 2.10, Pearson et al. investigated the formation of micelles from copolymer of 2-methacrylamido glucopyranose (MAG) and 5 -O-methacryloyl

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BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

FIGURE 2.8 Synthesis of SPCL–PLAMA copolymers.

SYNTHESIS OF BLOCK GLYCO-COPOLYMERS

129

TABLE 2.2 Molecular Weight and Molecular Weight Distribution Index of Star-Shaped SPCL-PLAMA Block Copolymers Synthesized by Direct ATRP of LAMA Monomer in NMP Solution at Room Temperature [LAMA]/ [SPCL-Br]

Entrya SPCL15 –PLAMA3 e SPCL15 –PLAMA7 e SPCL15 –PLAMA11 e SPCL75 –PLAMA5 f SPCL75 –PLAMA11 f

60 120 240 60 120

LAMA Conv. f PCL /f PLAMA b % %/% Mn ,GPC c Mw /Mn c Mn ,NMR d 20.0 23.3 18.3 33.3 36.7

54.2/45.8 33.7/66.3 24.8/75.2 78.5/21.5 62.5/37.5

25,030 30,200 — 52,100 60,500

1.21 1.18 — 1.33 1.29

12,860 20,370 32,540 43,970 55,230

a

The subscript numbers represent the repeating units of polymers. f denotes the weight fractions of PCL and/or PLAMA within block copolymers, which was determined by 1 H NMR. c Mw = Mn denotes the polydispersity index of the polymer, where weight-average molecular weight (Mw ) and number-average molecular weight (Mn ) were determined by GPC in DMF solution (polystyrene used as the calibration standard). d M n;NMR was determined from the integral ratio of the signal on the main chain of PCL ( CH2 , 2.20–2.29 ppm) and the signal on the main chain of PLAMA ( CH2 , 1.83–1.89 ppm) from the 1H NMRspectra. e [SPCL15 -Br]:[CuBr]:[Bipy]•1:1:2 and the polymerization time = 24 h. f [SPCL75 -Br]:[CuBr]:[Bipy]•1:1.5:3 and the polymerization time = 48 h. Source: Reproduced with permission from [37]. b

(c)

(d)

FIGURE 2.9 Transmission electron microscopy (TEM) micrographs of self-assembled aggregates from SPCL75 –PLAMA11 .

FIGURE 2.10 Synthesis of copolymer from MAG and MAU.

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BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

(a)

(b)

(c)

(d)

FIGURE 2.11 TEM images of micelles formed from: (a) PMAG59 -b-PMAU36 , (b) PMAG59 -b-PMAU55 , (c) PMAG59 -b-PMAU67 , and (d) PMAG55 -b-PMAU97 .

uridine (MAU) obtained by RAFT polymerization [42]. A bimodal molecular weight distribution was observed by them when they grafted MAU block to the PMAG, which was served as a macro-RAFT agent. These amphiphilic copolymers could self-assemble to micelles and with the increase of hydrophobic PMAU block lengths an increased tendency to form rods was observed (Fig. 2.11). 2.2.4 Other Methods Other methods such as polymer analogous reaction and click chemistry have also been used for the synthesis of glyco-copolymers. Click chemistry has recently been interested for its extensive applications in synthesis and modification of polymers [43–47]. The click chemistry has some advantages such as mild reaction conditions, highly tolerant to functional groups and high efficiency. At the same time, because of these features, click chemistry is very suitable for preparation of saccharidecontaining materials [48–50]. Xu et al. synthesized a series of biodegradable glyco-copolymer based on PCL [51]. The PCL was obtained by ring-opening polymerization (ROP) and various pendent saccharides were then attached onto the PCL backbone by click chemistry. As

SYNTHESIS OF BLOCK GLYCO-COPOLYMERS

131

FIGURE 2.12 Synthetic route of (a) the amphiphilic diblock glycopolymers and (b) the alkynyl sugars used for click chemistry.

shown in Figure 2.12a, PCL was used to initiate the ROP of 2-bromo-ε-caprolactone (BrCL) to get a diblock copolymer (PCL-b-PBrCL). The bromine groups in the PBrCL block were converted to azide groups by the reaction of the block copolymers with sodium azide. Subsequently, click chemistry between alkynyl saccharides (Fig. 2.12b) and the pendent azide groups of PCL-b-PBrCL gave out the glyco-copolymers. The saccharide content could be easily adjusted by the feed ratio of the alkynyl saccharides to the PCL-b-PBrCL. Meanwhile, this method is somehow versatile for displaying different type of saccharides. You and Schlaad reported an easy method for construction of amphiphilic glycocopolymers under mild conditions, which is different from click chemistry and is carried out without transition metal ions [52]. It is achieved by free radical addition of a 1-thioglucose derivative onto a 1,2-polybutadiene-based block copolymer. You and Schlaad started from 1,2-polybutadiene85 -block-polystyrene351 [(1) in Fig. 2.13, the subscripts denoting the average number of repeating units], which was prepared by sequential anionic polymerization of 1,3-butadiene and styrene in tetrahydrofuran (THF) [52]. They got a very low polydispersity index of the polymer as 1.15. After that 1,2,3,4,6-tetra-O-acetyl-␤-d-1-thioglucopyranose (see Fig. 2.13 (2)) and azoisobutyronitrile (AIBN) (molar ratio: [C=C]/[SH]/[AIBN] = 1:6:0.33) were added to the polymer in dry THF and the reaction mixture was stirred for 5 h under irradiation with a mercury lamp to yield polymer 3a. The gravimetric yield was nearly quantitative.

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BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

FIGURE 2.13 Synthesis of a glycopolymer through the radical addition pathway (Ac = acetyl).

2.3 pH-SENSITIVE GLYCOPOLYMERS Qiu et al. synthesized a special star-shaped polypeptide/glycopolymer copolymer from poly(␥ -benzyl-l-glutamate) and poly(d-gluconamidoethyl methacrylate) by the combination of ring-opening polymerization of ␥ -benzyl-l-glutamate Ncarboxyanhydride (BLG–NCA) and atom transfer radical polymerization of unprotected GAMA [53]. As shown in Figure 2.14, the ring-opening polymerization of BLG–NCA was initiated from star-shaped polyamidoamine (PAMAM) with four terminal amine groups. Then, the star-shaped PBLG (SPBLG) with terminal amine groups was converted into ATRP macroinitiator by functionalization with 2-bromo-2methylpropionyl bromide. After that, star-shaped poly(␥ -benzyl l-glutamate)-bpoly(d-gluconamidoethyl methacrylate) (SPBLG–PGAMA) was synthesized by ATRP of GAMA. The copolymer showed controlled molecular weight and low

133

O

O

N

O

GAMA, ATRP

OH

CH3 H2C C O O OH OH NH

NH2

HN

O

O

NH

NH2

O

O

SPBLG-PGAMA

O

O

BLG-NCA, ROP

O

NH

H 2N

H2N

SPBLG-NH2

NH2

O

NH

PBLG

O

O

(

O CH3 Br CH3

)

ET3N, CHCl3

Br

HO HO

SPBLG-Br

O PGAMA

OH

CH3 CH3 C C ( CH2 C ) Br O CH3 O OH OH NH

O

Br(H3C)2COCHN

Br(H3C)2COCHN

NHCOC(CH3)2Br

NHCOC(CH3)2Br

FIGURE 2.14 Synthesis of star-shaped SPBLG–PGAMA biohybrids via the combination of ROP of BLG–NCA and direct ATRP of unprotected GAMA glycomonomer.

HO HO

N

G0-PAMAN

NH

NH

H 2N

H 2N

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BLOCK GLYCOPOLYMERS AND THEIR SELF-ASSEMBLY PROPERTIES

polydispersity and the PBLG segment exhibited a helix structure. This amphiphilic block copolymer can self-assemble into aggregates in aqueous solution. The critical aggregation concentration (cac) value of this SPBLG–PGAMA copolymer was in the range of 1.67–4.61 × 102 mg/mL and the cac increased slightly with the increase of the composition of hydrophilic PGAMA block in the block copolymers. The cac value of star-shaped copolymer was apparently lower than that of the linear one, which indicates that the self-assembled aggregates of star-shaped copolymer are more stable than those of linear copolymer in aqueous solution. The authors also argue that the star-shaped SPBLG–PGAMA copolymers are more suitable for the drug delivery system because of the longer blood circulation time than linear copolymer. This star-shaped glyco-copolymers showed pH-sensitive self-assembly behavior, and the average size of the nanoparticles decreased gradually with the aqueous pH value. As shown in Figure 2.15, SPBL–PGAMA block copolymer with 36 repeating units of hydrophobic polypeptide block and long PGAMA segment (SPBLG36 –PGAMA39 , PGAMA = 59.4 wt%) aggregated into spherical micellar structure with an average diameter of 274.3 ± 7.0 nm. Interestingly, these glycopolymer-containing aggregates were much bigger than normal polymeric micelles obtained from linear polymers, which are usually less than 100 nm in diameter [54, 55]. The strong hydrogen-bonding interactions between these glycopolymershelled micelles, which cause the further aggregation of simple core–shell micelles, may be ascribed to the large size aggregates. As can be seen in Figure 2.16, the average size of the self-assembled aggregates decreased with the increase of pH value. Moreover, there is a critical change in particle size between pH 5 and 6, and the nanoparticle size at pH 5 was about five times larger than that at pH 6. This can be attributed to that the coil–helix transition

(a)

FIGURE 2.15 TEM photographs of the self-assembled aggregates from SPBLG–PGAMA copolymers SPBL G36 –PGAMA39 .

pH-SENSITIVE GLYCOPOLYMERS

135

FIGURE 2.16 Average size of deprotected SPLG39 –PGAMA7 nanoparticles as a function of pH at room temperature.

of PLG, which usually occurs at pH of 5.2, as the intermolecular hydrogen-bonding within the helical poly(l-glutamate) segments increased greatly at this pH [56]. This was also proved by proton nuclear magnetic resonance (1 H-NMR) spectra of SPLG–PGAMA at different pH values (Fig. 2.17). The signals of PLG segments were much weaker in acidic solution (pH = 5) than those in pH 10 due to the decreased

FIGURE 2.17 1 H-NMR spectra of SPLG–PGAMA in acidic (a, pH = 5) and basic (b, pH = 10) solutions.

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mobility of hydrogen-bonding associated with PLG segments and the shielding effect of PGAMA shell to the PLG core. 2.4 TEMPERATURE-SENSITIVE GLYCOPOLYMERS Zhang et al. synthesized a thermoresponsive block copolymer, poly(acryloyl glucosamine)-block-poly(N-isopropylacryamide) (PAGA180 -b-PNIPAM350 ), by RAFT polymerization [57]. This copolymer could simultaneously self-assemble and crosslink in aqueous medium forming core-crosslinked micelles, containing glycopolymer corona and PNIPAM stimuli-responsive core. They crosslinked this copolymer by acetal-type crosslinking agent, 3,9-divinyl-2,4,8,10-tetraoxaspiro undecane and resulted in a core–shell structure that is stable at high pH values, while the micelles readily decomposed in an acidic environment (Fig. 2.18).

FIGURE 2.18 Synthesis and crosslinking of (a) PAGA-b-PNIPAM and (b) the thermal responsive property.

TEMPERATURE-SENSITIVE GLYCOPOLYMERS

137

90 Hydrodynamic diameter (nm)

80 70 60 50 40 30 20 10

PAGA180 b PNiPAAm350 Crosslinked PAGA180 b PNiPAAm350

0 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 Temperature (°C)

FIGURE 2.19 Particle size as a function of temperature for linear () and crosslinked (◦) PAGA180 -b-PNIPAM350 .

This copolymer, PAGA-b-PNIPAAM, is water soluble up to a temperature of 28.8◦ C, which is below the lower critical solution temperature (LCST) of PNIPAm. The dissolved copolymers aggregate at pH = 7 and exhibit a hydrodynamic diameter of 20 nm. Above this temperature, the hydrodynamic diameter increases to about 90 nm, which is due to the extension of PNIPAM segments in the copolymer (Fig. 2.19). Moreover, this process is fully reversible over cooling/heating cycles and the hydrodynamic size remains roughly constant for both of the states. However, after crosslinking, the aggregates showed a more stable structure than the uncrosslinked copolymer. Interestingly, this crosslinked copolymer micelles can be hydrolyzed into well-defined free block copolymers at lower pH (Fig. 2.20, hydrolyzed in 12 h at pH 4 and in 30 min at pH 2). This property makes this crosslinking copolymer very

FIGURE 2.20 Hydrodynamic diameter as a function of degradation time of crosslinked PAGA-b-PNIPAM, concentration is 1 g/L, pH = 4.

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promising in drug delivery in which the decomposition of the drug containers under specific conditions would lead to a faster drug release and an easier clearance of the polymer.

2.5 CONCLUSIONS AND FUTURE TRENDS A great number of block glyco-copolymers have been successfully synthesized by various polymerization methods such as ATRP and RAFT. The chain length and chain structure can be well controlled by the advantages of modern polymerization techniques. With the different carbohydrates displayed on them, these block glycopolymers have been applied as biosensors, scaffold materials for tissue engineering, affinity matrix for protein recognition and pathogen capture, and drug/gene delivery devices. Block glyco-polymers with stimuli-responsive blocks that can change their physical and/or chemical properties under different conditions will receive intensive attention. Efforts will focus on the controlled synthesis and applications of these stimuli-responsive block glyco-polymers especially in biomedical fields.

ACKNOWLEDGMENT Q. Yang gratefully thanks help and support from Pro. Dr. Mathias Ulbricht.

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CHAPTER 3

CATIONIC GLYCOPOLYMERS MARYA AHMED and RAVIN NARAIN Department of Chemical and Materials Engineering and Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, Canada

3.1 3.2 3.3 3.4 3.5

Introduction Preparation of Cationic Polymers Complexation with DNA Biological Applications Conclusion and Future Directions References

143 146 155 160 161 162

3.1 INTRODUCTION The combination of synthetic polymers with natural macromolecules such as nucleic acids, proteins, and polysaccharides has been explored as an important field of research because these synthetic and natural macromolecules differ in many fundamental aspects. Such a combination of polymers with naturally occurring macromolecules is referred to as polymer bioconjugates. With the recent advancements in the field of nanotechnology, polymer bioconjugates have gained enormous attention in biomedicines and in material science [1]. The growing need for the successful synthesis of an ideal nonviral gene delivery vehicle has put great emphasis on designing the novel vectors to overcome the intercellular and extracellular barriers encountered during the gene delivery process. For this reason, various cationic polymers have been synthesized and their biochemical properties have been studied [2]. Polymer-mediated gene delivery systems have recently been developed as an alternative to viral-based transfection systems since polymers induce less immune and inflammatory responses, have a lower cost of synthesis, and possess a larger nucleic acid storage capacity. Although, these synthetic systems show great promise toward Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

143

144

CATIONIC GLYCOPOLYMERS

successful gene therapy, difficulties with low efficacy and high toxicity limit their use in clinical applications [3]. The successful development of nucleic acid therapies requires the synthesis of effective nonviral vectors for cellular delivery of the cargo. The biocompatibility of the delivery vehicle is largely dependent upon the stability of binding between the nucleic acid and the vector. The cellular uptake, endosomal release, and intracellular trafficking of the nucleic acid are some of the competent steps that can prematurely destabilize the complex binding and delivery [4]. In general, cationic polymers interact with the anionic molecules such as DNA (deoxyribonucleic acid) through electrostatic interactions to form nanometer-scale aggregates called polyplexes. The formation of polyplexes is an entropically driven process and is useful for the delivery of DNA to the cells for various reasons. The use of cationic polymers for DNA delivery purposes provides a macromolecular scaffold to overcome the intracellular barriers to gene delivery. Cellular uptake of free DNA by plasma membrane is usually inhibited by the larger size and negative charge of the macromolecule and to promote the entry of bare DNA inside the cell clinically limited methods such as electroporation or a gene gun has been used. In addition, once inside the cell its systemic regulation is inhibited by nuclease degradation. The complexation of anionic DNA with cationic polymers leads to the neutralization of charge on both components, thus reducing the toxicity of the vehicle and improving the efficacy of gene delivery simultaneously. The successful complexation of plasmid with polymeric vehicles depends upon many factors that will be discussed in detail in this chapter [2]. Cellular uptake of nonviral gene delivery vehicles predominantly occurs through electrostatic interaction between cationic polymer–DNA complexes and anionic cell surface proteoglycans, regardless of the specificity of the vehicle. This mechanism of interaction between polyplexes and proteoglycans has been confirmed, as the removal of glucosaminoglycans from the cell surface shows inhibited gene transfer by poly(l-lysine) (PLL) and DNA complexes [5, 6]. The study of mechanism of uptake of cationic polymer–DNA complexes is crucial for successful gene transfer and is found to proceed through various endocytic routes. Polyethylenimine (PEI) and DNA complexes are found to be engulfed by phagocytosis, as the process was inhibited by the staurosporine, an inhibitor of protein kinase C (PKC), which upon phosphorylation activates phagocytosis [7]. In contrast, uptake of PLL–DNA complexes in Hep G2 cells is found to proceed by clathrin-mediated endocytosis, thus leading to successful gene transfer to the host cells [8]. The cationic vehicles are known to undergo “enforced” endocytosis due to the electrostatic interactions between the vehicle and the host cells, thus contributing toward the higher chances of successful gene transfer, regardless of the size of vehicle. The cytosol presents multiple barriers in gene delivery, after the internalization of complexes and their release inside the cells. The mobility of free plasmid inside the cytoplasm along with fragmentation of free DNA in cytosol are inherent problems in the transfer of free DNA to the nucleus [4]. The cationic polymer-based vehicles compact DNA into smaller particles, thus protecting the DNA from degradation in the cytoplasm, and aiding the movement of DNA into the nucleus. Nuclear entry of the plasmid DNA is strictly regulated by the nuclear envelope. To gain access to

INTRODUCTION

145

the transcriptional machinery of the nucleus of dividing cells, plasmid DNA enters the nucleus during cell division. Transfection studies with cationic vector–DNA complex shows significantly higher levels of gene expression than that of free plasmid, suggesting that a positively charged vector can possibly exert a nuclear localizing effect, thus promoting the movement of polyplexes inside the nucleus [9]. Although, cationic DNA carrier systems are a preferable source of gene delivery in vitro, compared to the efficacy of transfection of free plasmid, the systemic delivery is limited by the instability of polyplexes in physiological conditions. The cationic polyplexes lead to vascular blockage due to the aggregation of complexes at higher salt concentrations (150 mM). They can also bind to serum proteins, thus encouraging phagocytosis and rapid clearance of complexes from the system. Another issue related to polyplex-mediated transfection is the need for cationic polymers to ultimately release DNA, once it has transported the DNA into the cytoplasm or in the nucleus of a cell [10]. At this point the cationic component of a polyplex is no longer required and can actually hinder the transfection process by limiting the access of DNA to cellular machinery. The experimental evidence suggests that “DNA unpackaging” is a relatively inefficient process [2]. The incorporation of new designs of elements into the structures of cationic polymers has resulted in a remarkable progress to improve the functions of cationic polymer-based gene delivery vehicles. The modifications incorporated into basic cationic polymers are focused to improve their cytotoxicity, to reduce aggregation, to induce sponge effect, and to incorporate targeting agents to achieve specificity [11–14]. The most common modification of cationic polymers is their PEGylation(incorporation of poly(ethyleneglycol) into polymers structure, which is found to reduce the toxicity and aggregation of polyplexes, and increase their circulation time in blood, but it severely limits the transfection process itself by decreasing the surface interaction of polyplexes with cells and by causing the premature dissociation of DNA from polymeric vehicles [10]. The need is to create novel synthetic polymeric gene delivery scaffolds that lack the drawback of PEG-modified vehicles. The major biological communication events exploit the structural and functional diversity of glycoconjugates. Early events in the infectious cycles of many microorganism and metastatic processes involve carbohydrate-mediated recognitions of host by pathogens. The glycoconjugate-based selectively small molecules provide the potential to control these events. The weak affinity of saccharide ligands towards their receptors, showing association constant beyond 106 M−1 is a major drawback of carbohydrates for the in vivo usage. The higher affinity is typically obtained by the cluster glycoside effect. The cluster glycoside effect is attributed to the high affinity of multimeric carbohydrates toward the targeted ligand, which is ideally obtained by the advent of glycoconjugates [15, 16]. Despite of the inherent challenges attributed to the construction of glycoconjugates, the main driving force toward the rapid advancement of the field of glycopolymers is the transfer of biological functions of carbohydrates toward synthetic polymers for biomedical, pharmacological, and cosmetics applications. The glycopolymers of natural and synthetic origin have been widely used in biomedicines for gene and drug delivery processes, due to the characteristic interactions of carbohydrate moieties with living systems.

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Chitosan, a naturally occurring cationic polysaccharide, has been used for gene delivery purposes. These and other carbohydrate-based gene delivery vehicles are noncytotoxic but reveal low gene delivery efficiency in many cell lines [17–19]. The dominant reason for low gene transferability of naturally occurring cationic polymers is found to be their low molecular weight, which also contributes toward their low DNA condensation ability [20]. Schizophyllan, a polysaccharide with antitumor effects, is an example of a noncationic glycopolymer that binds with oligonucleotides via hydrogen bonding between the bases of DNA and single chains of the polymer. This naturally occurring carbohydrate was used in vitro to promote the delivery of oligonucleotides in mammalian cells. The drawback of a schizophyllan-based gene delivery vehicle is that DNA must possess a poly A tail or poly C tail as an only homonucleotide sequence to bind with macromolecule [21, 22]. Another approach toward the construction of cationic glycopolymers for gene delivery purposes is the postmodification of cationic polymers with carbohydrate-based macromolecules. For example, the incorporation of ␤-cyclodextrin, a cyclic oligosaccharide, into cationic polymers shows transfection levels much higher than those of PEI and lipofectamine, accompanied by low cytotoxicity in the presence of serum. However, in vivo studies show that these polymers suffer from aggregation at high ionic strength. The PEGylation of the vehicle is found to overcome the aggregation problem along with the reduction in charge density of the cationic polymer [23–25]. The diethylaminoethyl dextran (DEAE–dextran) is a commonly used dextran-based cationic polymer used for gene transfer. Recently, dextran-spermine-based polymers have been synthesized that rival commercially available gene delivery vehicles. The PEGylated spermine dextran conjugates have been used for in vivo delivery of DNA and show successful gene transfer, when administered intravenously at higher polymer-to-DNA weight ratios [26, 27]. The libraries of synthetic polymers are larger than natural ones and can be produced at a much cheaper rate. On the other hand the molecular binding blocks of nature are interesting because of their exceptional tendency to self-organize. Since both categories of biological and synthetic polymers are quite broad, the construction of these hybrid molecules is fast growing and constantly changing the field of chemistry. Reineke and co-workers have combined the successful transfection efficiencies of PEI with chitosan, resulting in polymers with carbohydrate moieties along a linear amine backbone [3, 28]. The synthetic cationic glycopolymers produced are then used for the investigation of transfection ability as a function of type of carbohydrate monomer used, number of amine groups present on the polymer, and the stereochemistry of hydroxyl groups of the carbohydrates [3, 28, 29]. The next section briefly discusses the synthetic strategies employed to produce cationic glycopolymers by using several polymerization techniques.

3.2 PREPARATION OF CATIONIC POLYMERS Amine-based polymers have been exploited for several biomedical applications, for instance, gene and drug delivery, and as scaffolds for cultured cells. However, their

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PREPARATION OF CATIONIC POLYMERS

preparation is extremely challenging. The ring-opening polymerization is one of the most commonly used methods, which has been employed in the past to produce commercially available cationic polymers, including polyethylenimine (PEI) and poly(l-lysine) (PLL) [30, 31]. PEI is one of the prominent examples of cationic polymers capable of producing higher gene levels during in vitro studies [31]. The transfection efficiencies of PEI are found to be directly related to its molecular weight. The increase in molecular weight produces a direct increase in transfection efficiency of the vehicle accompanied by higher cytotoxicity. The optimum range of molecular weights required for higher transfection efficiencies is found to be in the range of 5–25 kDa [31]. The linear structure of higher molecular weight PEI (LPEI) is simply obtained by acid hydrolysis of commercially available poly(2-ethyl-2-oxazoline) of 50-kDa molecular weight under reflux. The LPEI of different molecular weights were obtained by ring-opening polymerization of 2-ethyl-2-oxazoline (EtOXZ) followed by acid hydrolysis. The polymerization of EtOXZ is initiated by methyl-ptoluenesulfonate (TSOMe) in acetonitrile at 80◦ C and the degree of polymerization is estimated by proton nuclear magnetic resonance (1 H-NMR) spectroscopy. The acid hydrolysis of the poly(2-ethyl-2-oxazoline) (PEtOXZ) was carried out as a deprotecting step with the excess hydrochloric acid (HCl), thus yielding LPEI polymers of different molecular weights [31] (Scheme 3.1). As discussed earlier, the synthesis of cationic glycopolymers is usually achieved by the postmodification of a cationic polymer with naturally occurring polysaccharides. The synthetic polymers with pendant saccharide moieties have received enormous attention in the biochemical and biomedical fields. Glycopolymers are hydrophilic water-soluble, biocompatible molecules that are potentially bioactive toward specific biomolecules, depending on the nature of saccharide moieties. For the extended

C2H5

O O

N

+

S OMe

H3C

O 2-EtOXZ

CH3CN

N n-1 N

O

+

TsO -

PEtOXZ O

TsOMe

+ H3O, excess

H *

N n

OH

Linear PEI

SCHEME 3.1 Synthesis of linear structures of PEI, via ring-opening polymerization of 2-ethyl-2-oxazoline (EtOXZ), followed by acid hydrolysis as a deprotection step. (Adapted from Ref. [31].)

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CATIONIC GLYCOPOLYMERS

O

O O

O

O OH

O OH

Cl O

O tBuOK,

O

DMF O

O O Diisopropylidene-D-glucose

Diisopropylidene-D-glucose vinyl ether

SCHEME 3.2 Synthesis of sugar monomers (glucofuranose vinyl ether, iPGFVE) for cationic polymerization. (Adapted from Ref. [33].)

applications in the biomedical field, proper control on the molecular weight and molecular weight distribution of the glycopolymers is required [32]. Chemical synthesis of sugar-containing polymers is challenging due to the presence of multiple hydroxyl groups in sugar with nearly identical reactivities, which poses a challenge in achieving the selective modification and polymerization [16, 33]. High-yield glycosylation methods and advanced protection chemistries are usually used to overcome the above-mentioned problem [33]. The synthesis of glycopolymers is achieved by both ionic polymerization and controlled radical polymerization techniques. The key step to polymer synthesis is the synthesis of pure vinyl sugar monomers, which is usually obtained by coupling an unsaturated hydrocarbon to the carbohydrate molecules by ether, amide, or ester linkage as shown in Scheme 3.2 [33]. Cationic polymerization requires the use of monomers that do not undergo any reactive functions, thus modified sugars with hydroxyl-protected groups were modified with unsaturated hydrocarbon, before subjecting them to polymerization, as shown in Scheme 3.3. A diisopropylidene glycosidic vinyl ether monomer (glucofuranose vinyl ether, iPGFVE) was prepared by mixing the solution of 1,2:5,6-diisoprpylidened-glucose, crown ether, and 2-chloroethyl vinyl ether in dry dimethylformamide (DMF) at 80◦ C for 18 h, as shown in Scheme 3.3. The polymerization of iPGFVE was triggered by ZnCl2 in the presence of the macroinitiator polystyrene acetal. The cationic glycopolymers poly(iPGFVE) of various lengths were obtained followed by the deprotection step of furanose units by the treatment of copolymer in water–trifluoroacetic acid (1/9 v/v mixture) for 1 h [34]. The block copolymerization of polystyrene (PS) and poly(iPGFVE) was further carried out under the same conditions. To prevent the excessive steps of protection and deprotection of glycomonomers during polymerization, living radical polymerization was found to be an emerging technique for the polymerization of glycomonomers. The advantage of the living radical polymerization technique is that the reactions can be run in aqueous conditions on unprotected sugar monomers.

PREPARATION OF CATIONIC POLYMERS

149

I OCH2CH3

n -1

m

+

O

O

iPGFVE

ZnCl2 CH3OH/NH4OH

n -1

O

O

O

O

OCH2CH3 m-1

iPGFVE

OMe

iPGFVE

SCHEME 3.3 Cationic polymerization of glucofuranose vinyl ether (iPGFVE), using polystyrene acetal as a macroinitiator. (Adapted from Ref. [34].)

The olefin-based ring-opening metathesis polymerization (ROMP) reactions are found promising toward the synthesis of a new class of sugar-substituted polymers as the reaction should tolerate highly polar monomers bearing unprotected sugars. For this purpose, a sugar monomer (7-oxanorbornene derivative) was first produced by attaching the protected carbohydrate to the oxanorborene skeleton via C-glycoside linkage, followed by the removal of triethylsilyl protecting groups, yielding unprotected glycomonomer. The ROMP of glycomonomer was then conducted by treatment with ruthenium trichloride (RuCl3 ) in water, as shown in Scheme 3.4. The high yield of the glycopolymer was obtained from this reaction [35]. It was later reported that the metathesis polymerization initiated by RuCl3 is not living as the polymers obtained are of high polydispersity and lack the molecular weight specificity. The ruthenium carbine catalysts are found to promote the living polymerizations of strained monomers such as norbene and cyclobutene with exceptional tolerance of functionality. For this reason glucose monomers were modified with norbene via an amide linkage. These monomers were then polymerized in the presence of protecting group chemistry. The procedure was further carried out to synthesize block copolymer under the same conditions [36]. Nitric-oxide-mediated living free radical polymerization (NOMP) allows the polymerization of wide varieties of monomer families with accurate control over molecular weight and polydispersities. These systems are based on ␣-hydrido nitroxides and have the advantage of facile introduction of functional groups into the initiator structure with standard organic synthesis techniques. The method was used

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CATIONIC GLYCOPOLYMERS

O OTES

O

O

O O SETO

OTES

O H

OH

+ N ICH3

H

O

Cl

O O

O

DMAP, NPr 3, CH2Cl2 HF, pyridine

HO

OTES HO O-triethylsilyl-D-glucopyranoside

HO

OH

O

HO

O

OH OH

CH2OH

RuCl3, H2O

O n H

H

O

O O

O

HO HO

HO CH2OH

O

HO

O

OH OH OH

SCHEME 3.4 Ring-opening metathesis polymerization of unprotected glycomonomer producing polyglycomers in high yield. (Adapted from Ref. [35].)

to synthesize complex lipid-based amphiphilic glycopolymers using, alkoxyamine initiator as shown in Scheme 3.5. For example, the initiator 2,2,5-trimethyl-3{1-[4-di(octadecyl)aminomethyl]phenylethoxy}-4-phenyl-3-azahexane was used to polymerize poly[1, 2, 5, 6-di(isopropylidene)-d-glucose-2-propenoate], a structure obtained after the protection of hydroxyl groups of d-glucose-2-propenoate by isopropylidene. The polymerization was conducted at 125◦ C under argon in the presence of initiator and 0.05% of the corresponding nitroxide. However, the sugar residues were found to undergo partial decomposition at this elevated temperature. To overcome these problems, the reaction was carried out in DMF at 105◦ C. The choice of DMF was governed by the insolubility of glucose monomers in other traditionally used solvents for NOMP such as chlorobenzene and xylene. However, an increase in the amount of DMF above 30 wt% leads to the decrease in the rate of polymerization and increase in polydispersity. The deprotection of the lipoglycopolymer was further obtained in an acidic environment [35]. The procedure was further modified to produce water-soluble homo and block glycopolymers and is called (2,2,6,6-tetramethylpiperidinyl-1-oxy) TEMPO-mediated free radical polymerization [38, 39]. The ability of polymer chemists to successfully control the parameters of synthetic macromolecules such as molar mass, molar mass distribution, and chain

PREPARATION OF CATIONIC POLYMERS

O

151

N

+

O

O n

O O

O O

O CH3(CH2)17

N (CH2)17CH3

O

N

O

N

n

O

O CH3(CH2)17 H n

O

OH HO

N

O O

O

(CH2)17CH3

O HO

CH3(CH2)17

N

O

O

O OH

(CH2)17CH3

SCHEME 3.5 Synthesis of lipo-glycopolymer by nitric-oxide-mediated living free radical polymerization. (Adapted from Ref. [37].)

end-functionality is highly attributed to the advent of free radical polymerization. The technique is extremely versatile and facilitates the controlled copolymerization of a large number of olefinic monomers. Typically, glycopolymers prepared by this polymerization technique were of protected precursors followed by postpolymerization deprotection, as discussed earlier. Lowe et al. and others have shown the synthesis of homopolymers of protected glycomonomers and of their block copolymers by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protected glycomonomers were polymerized in controlled fashion employing the 4-cyanopentanoic acid dithiobenzoate/4,4 -azo-bis(4cyanopentanoic acid), CTA–initiator combination in the water–ethanol mixture at

152

CATIONIC GLYCOPOLYMERS

Ph

S S O

O O

S

AIBN, DMF, 60°C X = Ph or CN

O O

O

S

N

O O

AIBN, DMF, 60°C

O

O

O

O

S X O

O

O O

O

X

X

O O

O

O

S

N

Ph

S O

O O

O

O

Ph TFA/H2O, 5:1 X RT, 1h

O

O

O

OH OH

HO

O

S

N

HO

SCHEME 3.6 RAFT polymerization of cationic glycopolymer (PMAGP-b-PDMAEMA). (Adapted from Ref. [40].)

70◦ C [40, 41]. For example, methyl-6-O-methacyloyl-1,2:3,4-di-O-isopropylidened-galactopyranose (MAIpGP), a protected glycomonomer was polymerized by RAFT in DMF utilizing cyanoisopropyl dithiobenzoate (CDB) and 1-cyano-1-methylethyl dithiobenzoate (CMED) as RAFT agents. This methacrylic glycomonomer was polymerized in controlled fashion yielding 3-O-methacryloyl-d-galactopyranose (PMAGP). The block copolymers of PMAGP were further obtained, with 2-(dimethyl amino) ethyl methacrylate (DMAEMA), followed by their deprotection in acidic environment [41] (Scheme 3.6). The researchers are now exploring the feasibility of producing glycopolymers in the absence of protected groups. Narain and Armes first reported the direct strategy to synthesize glycopolymers via ATRP, without the protection of hydroxyl groups of carbohydrates. The synthesis of sugar-based monomers and polymers is usually achieved by masking the secondary alcohols to ensure that monofunctional monomers are obtained, as shown above, but the process is time consuming and costly. One possible approach is to exploit the differences in reactivity between the primary and secondary alcohols in sugar monomers, but the facile functionalization of vinyl sugars without protecting group chemistry is a challenge. The tolerance of ATRP toward many functional groups suggests new possibilities for the direct synthesis of controlled-structure sugar polymers. The controlled structures of near monodisperse sugar-based polymers were prepared directly under mild conditions without protecting group chemistry. The target monomer 2-gluconamidoethylmethacrylate (GAMA) was synthesized in methanol at 20◦ C by the reaction of 2-aminoethyl methacrylate with d-gluconolactone. d-gluconolactone is a raw material obtained from the

PREPARATION OF CATIONIC POLYMERS

O O O

n

O

Me NH

O OH HO OH HO CH2OH

O

Br

n

O x

153

O O

O x O

Br

CuBr/bpy, 20°C PEGx -Br (x = 7, 23, 113) Water, methanol or water/methanol mixtures

NH O OH HO OH HO CH2 OH

SCHEME 3.7 Synthesis of homopolymer polyGAMA by ATRP. (Adapted from Ref. [42].)

oxidation of d-glucose. Ring opening of the d-gluconolactone occurs in the presence of triethylamine, which reacts with HCl to generate the free primary amine in situ, as shown in Scheme 3.7. This synthetic approach leads to monofunctional GAMA with no side reactions and after 5 h at 20◦ C GAMA was isolated by precipitation in isopropanol. The polymerization of GAMA was then obtained using PEG-based ATRP initiators, in combination with a Cu(I)Br/2bpy catalyst. The polymerization was achieved by dissolving the monomer in methanol or water–methanol mixture, prior to the addition of initiator and catalyst. The target degree of polymerization was varied from 30 to 100 and polymers were produced of 8- to 10-kDa molecular weight. The diluted reaction solutions were treated with alumina to remove the copper catalyst and purified GAMA homopolymer was obtained with 65–80% yield. Although, the water–methanol mixture significantly improved the rate of reaction, the polydispersities of polymer were found to increase proportionally with the increase in water content. The best living character was obtained in pure methanol, showing the slow reaction rate but the polymers produced were of narrow polydispersity (1.19). The block copolymer synthesis were then obtained by methanolic ATRP synthesis [42]. However, the presence of heavy-metal residues limits the use of these polymers for biomedical purposes. The RAFT process is proven to be the most versatile technique of radical polymerization for the monomers of choice and general polymerization conditions. RAFT can be employed to any monomer capable of undergoing free radical polymerization. The ideal tolerance of a variety of monomers and reaction conditions make RAFT ideal for the synthesis of glycopolymers; also this system offers a new synthetic technique to produce well-defined glycopolymers without the need of metal catalysts. First demonstration of glycopolymer synthesis by RAFT was provided by Lowe and Wang without the need of protection group chemistry [40]. Poly (2-methacryloxyethyl glucoside) was synthesized by RAFT in aqueous phase employing 4-cyanopentanoic acid dithiobenzoate (CTP) as chain transfer agent (CTA). Pseudo-first-order kinetics and linear relationship between molar masses and conversion were obtained. The

154

CATIONIC GLYCOPOLYMERS

O

O NH OH OH HO HO

CTP/ACVA H2O/DMF (5:1) 70°C

NC

S

NH

NH

OH OH

OH O HO HO

S CTP:

S n O

HO

CN S

COOH

NH

OH

O CN

ACVA:

N

HOOC

N

COOH

NC

SCHEME 3.8 RAFT Polymerization of 3-gluconaminopropyl methacrylamide (GAPMA) using CTP as chain transfer agent and ACVA as initiator. (Adapted from Ref. [32].)

block copolymers were further obtained with 3-sulfopropyl methacrylate (SPMA) with no detectable low-molecular-weight peak in the size exclusion chromatography (SEC); however, polydispersities increased and high-molecular-weight impurities were found [40]. This strategy was further modified and improvised by Deng et al. [32]. The facile one-step synthesis of glycomonomers 2-gluconamidoethyl methacrylamide (GAEMA) and 3-gluconamidopropyl methacrylamide (GAPMA) containing stable amide linkages, in the absence of protecting group chemistry, were shown by ring opening of sugar lactones. Well-defined glycopolymers were prepared by RAFT using CTP as CTA and 4,4 -azo-bis(4-cyanovaleric acid) (ACVA) as an initiator in aqueous solution at 70◦ C, as shown in Scheme 3.8. The clear indication of successful polymerization was obtained by the evolution of linear Mn . Pseudo-first-order kinetics, and close values of experimental Mn to theoretical Mn confirmed that polymerization is achieved in a controlled manner [32]. After establishing the appropriate experimental conditions for glycopolymer synthesis, the cationic glycopolymers were synthesized subsequently. For example, diblock copolymers of GAPMA-b-APMA and GAEMA-b-APMA were prepared by the RAFT via the macro-CTA technique. GAEMA and GAPMA were first homopolymerized as show above. The polymerization was allowed to proceed to conversion of 60–75% to ensure the higher efficiency of the macro-CTA. Considering the bulkiness of glycomonomers and of the glycopolymers lower DPn were targeted for the first block in the diblock synthesis. RAFT is highly effective technique toward the polymerization of methacrylamides and methacrylates-based monomers, the polymerization of aminoalkyl methacrylamides, namely, 2-aminoethyl methacrylamide (AEMA) and 3-aminopropyl methacrylamide (APMA) was obtained, with the amino groups in protonated form. The polymerization was carried out at 70◦ C, using ACVA as radical initiator and CTP as chain transfer agent [43] (Scheme 3.9). The diblock

COMPLEXATION WITH DNA

155

O CTA/ACVA O HN

S

Water/Dioxane (2:1) 70 °C

NH2.HCI

NC HN

O

S

NH2.HCI S

CTA:

n

HO

CN S

COOH

CN ACVA: HOOC

N

N

COOH

NC

SCHEME 3.9 RAFT polymerization of N-(2-aminoethyl) methacrylamide hydrochloride (AEMA). (Adapted from Ref. [43].)

copolymers constituting both amino groups and pendant sugar moieties were synthesized, and the amino groups of the polymers were further employed in bioconjugation reactions or complexation with DNA.

3.3 COMPLEXATION WITH DNA The cationic polymers have largely been used for gene delivery purposes in vitro due to their ability to condense and protect the gene of interest from degradation. Amine-containing polymers being positively charged at physiological pH, form ion pairs with DNA and have the potency to transfect several cell lines [44]. However, the major issues in this regard are the efficacy of transfection and cytotoxicity of the cationic gene delivery scaffold, during the gene transfer. The gene delivery for systemic applications requires the synthesis of monodisperse, small nanoparticles, capable of extravasation and nuclear translocation. The binding and compaction of the DNA by nonviral vectors is the first crucial step in this complex process and is found to play a dominant role in improving gene delivery efficacy [45]. It has been found that complexation of plasmid with DNA involves several factors, which upon consideration can significantly improve the role of these nonviral vectors. The study of biophysical characteristics of the interaction of DNA with several different cationic polymers reveal that the size of the DNA–polymer complex is dependent upon the physical properties of the polymer, rather than upon the size of DNA itself, and it is correlated to the molecular weight of the polymer. For example, higher molecular weight polymer (∼224 kDa) produces polyplexes of about 100–300 nm in diameter, while lower molecular weight polymers (∼4 kDa) yield complexes of 20–30 nm diameter. Although the morphology of the polymer–DNA

156

CATIONIC GLYCOPOLYMERS

complexes is independent of the type of polymer used, the aggregation behavior of the complexes is regulated by the polymer structures. For instance, clustering is common for less flexible structures such as intact dendrimers and PLL complexes [46]. The major difference between the cationic polymers with respect to the chemical structures is the type and relative number of protonatable amines. All the polymers possess primary amines that are predominantly protonated at neutral pH. For example, the acid–base titration curves of PEI exhibit considerable buffer capacity over entire pH range, while PLL shows comparatively lower buffering capacity below pH 8, thus indicating that general properties of these complexes will differ significantly from one another. The cationic polymers are found to demonstrate the maximum transfection activities at charge ratios with an excess of primary amines of cationic polymer to phosphate of DNA. The surface charges of the polyplexes are estimated by zeta potential (␨ potential) and strong positive ␨ potential is observed for the polyplexes showing significant transfection efficiencies. The classical colloid theory states that an important parameter to control the aggregation behavior of the complexes is the charge on the surface of the particle. Thus, polyplexes with strong ␨ potential are expected to show less aggregation, contributing toward higher transfection efficacy [46]. The ability of cationic polymers to bind and condense DNA into particles by varying the different parameters of cationic polymers, including charge density, order of cationic charge on the polymer surface, and the length of side chains, has been studied in detail using gel electrophoresis and fluorescence inhibition assay, and their physical properties have been characterized using atomic force microscopy (AFM) and ␨ potential. The principle of particle formation following cooperative binding of polycation to DNA, in the presence of different types of polycations, is explored and the following molecular parameters are investigated:

Distance of Cationic Charge from Polymer Backbone: Length of Side Chain The distance of cationic charges from the polymer backbone has an important influence on the properties of the complexes formed with DNA. The polymers with longer side chains tend to be less efficient at condensing DNA than polymers with shorter side chains, regardless of degree of polymerization. The polymers with charges closer to the backbone show efficient condensation of DNA [e.g., poly(vinyl amine) hydrochloride, pVA·HCl] and low Min fluorescence (F) and IF50 (inhibition of fluorescence) values (Min F is stated as lowest fluorescence level achieved at any Nitrogen/Phosphorus (N/P) ratio, and IF50 is N/P ratios of the polymer causing 50% fall in measured fluorescence). In contrast, polymers with long lateral side chains (poly[methacryloyl-Gly-Gly-NH-(CH2)6 -NH2 ] hydrochloride, pMADGHDA·HCl) showed poor condensation ability. The AFM images could not be obtained for the polyplexes formed with low-molecular-weight polymers such as pVA·HCl, as complexes tend to flocculate in water. On the other hand, cationic polymers with side chains of intermediate lengths were found to produce discrete particles of typical diameter of 100 nm. DNA complexes with longer side chains (pMADGHDA·HCl) were often loose with structural irregularities, and the DNA was found to be protruding from the complexes even several hours following the initial mixing of polymer.

COMPLEXATION WITH DNA

157

The resistance of polyplexes to anions was studied by estimating the interactions of cationic polyplexes with poly(l-aspartic acid) (pLAA) and restoration of ethidium bromide/DNA fluorescence (RFI50 ) was monitored. It was found that polymers with shorter chains (pVA·HCl) are most resistant to pLAA, when compared to the polymers with longer side chains. The surface charge of the complexes monitored by ␨ potential showed that the ␨ potential of pVA·HCl was found to be zero, owing to their tendency to flocculate in water. In contrast, polymers with medium molecular weights (40–80 kDa) or with long pendent chains produce complexes with positive ␨ potential, for example, pMADGHDA·HCl showed a potential of 36 mV. The transfection efficiency of polyplexes, produced with cationic polymers of varying side chain lengths, was then studied in vitro using 293 cells. It was found that polymers with short side chains (pVA·HCl) gave no significant spontaneous transfection, and slight increase in transfection efficiency compared to DNA alone is observed. On the other hand, complexes formed using pMADGHDA·HCl, despite the low molecular weight of the polymer, were found to mediate higher transfection levels, and the difference in transfection efficiencies of the polymers of varying chain length is attributed to their differences in net ␨ potential. Finally, the transcription activity of polyplexes was studied as a function of chain length by the direct inoculation of polyplexes in Xenopus oocytes. It was found that complexes-based on shorter chain lengths, although they exhibit poor transfection abilities, show significant gene expression during transcription; for example, pVA·HCl complexes showed over 60% gene expression, suggesting that despite their high stability to polyanions they are still suitable for intracellular transcription. The polymer with long side chain length pMADGHDA·HCl gave relatively low gene expressions.

Order of Cationic Charge or Influence of Charge Spacing along the Polymer Backbone To study the influence of charge spacing on the biophysical properties of polyplexes, a series of random copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA, uncharged polymer) with cationic quaternary amine containing polymer 2-(trimethylamino)ethyl methacrylate chloride (TMAEM·Cl) were synthesized with different ratios of TMAEM·Cl monomer (5, 15, 50, 75, and 100 mol% masses per charge) to HPMA monomer. It was found that, although complexes containing 50% pHPMA were unable to mediate 50% inhibition of EtBr/DNA fluorescence at any N/P ratio studied, the agarose gel electrophoresis showed that all copolymers synthesized including the one with 95% HPMA and with only 5% cationic character (1 positive charge is surrounded by 19 uncharged HPMA groups) are able to mediate agarose gel shift assay. In addition it was found that fluorescence of complexes with higher cationic component was lower and vice versa, indicating the strong binding of DNA with high cationic component, thus resulting in the formation of tighter complexes. AFM images showed that the polymers with the higher charge density of cationic component were capable of forming discrete structures, while extended structures were observed with cationic component of the polymer less than 50%. The positive ␨ potential was measured for all the polymers synthesized, supporting the agarose gel electrophoresis assay shift observed. The ␨ potential

158

CATIONIC GLYCOPOLYMERS

measured was in the range of 10–50 mV for all the polyplexes produced; the ␨ potential of free DNA is −80 mV. Although all the copolymers produced did show some transfection efficiencies in 293 cells, the levels were very low when compared to poly(l-lysine) (PLL·HBr) but were similar to those reported by pTMAEM·Cl homopolymer, and the similar results were obtained when the polymers were tested for their transcriptional activity. The results indicate that though charge spacing on the polymer backbone can significantly affect the physical properties of complexes formed by these polymers, their biological properties including transfection and transcription abilities remained unaltered, possibly due to their ability to condense DNA at all ratios of cationic polymer. pTMAEM·Cl (a cationic polymer bearing one quaternary charge per methacrylate-based monomer) was also compared to poly[1,3-bis-(trimethylamino)isopropyl methacrylate iodide] pBTMAIPM·I2 (a polymer bearing two quaternary positive charges/methacrylatebased monomer), thus creating a difference in charge densities of two similar polymer structures, and their biophysical properties were investigated. It was found that the increase in charge density had no significant effect on the properties of complex formation, and the same lower transfection values were observed for pBTMAIPM·I2 , despite the higher charge density of the polymer.

Influence of Charge Density along the Polymer Backbone The comparison of biophysical properties of similar molecular weights of primary, tertiary, and quaternary amine-based polymers, namely, (poly[methacryloyl-NH-(CH2 )2 NH2 ] hydrochloride) pMAEDA·HCl, (poly(2-dimethylaminoethyl methacrylamide), pDMAEMam, and (2-(trimethylamino) ethyl methacrylate chloride) pTMAEM·Cl, respectively, allowed the investigation of the influence of different charge densities on the properties of complexes formed. Quaternary charges were comparatively more efficient at DNA condensation, achieving low IF50 values. In contrast tertiary aminebased polymers (pDMAEMam) showed poor DNA condensation abilities and failed to decrease EtBr/DNA fluorescence to 50% of the starting value even at N/P ratio of 4. This poor ability of pDMAEMam to produce polyplexes may be due to its presence in basic form leading to lower levels of protonation in water than other polymers that were produced in salt form. AFM showed no difference in polyplexes formation with all methacrylate-based polymers, showing the formation of discrete complexes of about 100 nm diameter; also no trend was observed in the ␨ potential of polyplexes produced. Methacrylate-based polymers with primary (pMAEDA·HCl) and tertiary amine groups (pDMAEMam) showed higher transfection values that were comparable to PLL·HBr. On the other hand, complexes with quaternary amine groups (pTMAEM·Cl) showed much lower levels of transfection, and transfection was only marginally increased compared with the intrinsic transfection efficiency of free DNA. The evaluation of transcription activity indicated that transcription was higher for primary (77% higher than that of free DNA), and tertiary amine-based polymers (71% higher than that of free DNA). The quaternary amine-based polymer showed slightly less transcriptional activity (41% increase than that of free DNA), indicating that

COMPLEXATION WITH DNA

159

cellular transfection by these complexes may be subjected to particular problems of gaining access to cytoplasm and nucleus.

Effect of Molecular Weights of Polycations or DPn on Properties of Complexes Formed The formation of polymer DNA complexes by self-assembly as a function of molecular weight of the polymer was measured by the inhibition of EtBr/DNA fluorescence. The simplest methacrylate-based polymers pMAEDA·HCl showed that increase in molecular weight from 19 to 42 kDa improves DNA condensation ability of the polymer proportionally. The similar results were obtained using longer side chain polymer (pMAGEDA·HCl). However, the quaternary amine-based polymer (pTMAEM·Cl) showed no effect on DNA condensation ability as a function of molecular weight of the polymer. The complex formation of pTMAEM·Cl with DNA was studied in molecular weight range of 5–413 kDa, the polymer fractions of different molecular weights showed similar condensation abilities. The size of complexes formed with DNA was measured by AFM. It was found that the molecular weight of primary amine-based polymers did not affect the morphology of the polyplexes formed. However, tertiary and quaternary amine-based polymers (pMAGEDA·HCl and pMADGHDA·HCl) showed the presence of defined structures at higher molecular weights, in contrast to lower molecular weight polymers that yielded extended structures. pTMAEM·Cl also showed the significant effect of polymer molecular weight on structure formation, where low-molecularweight polymers produced extended structures, probably due to the aggregation of polyplexes. The transfection ability of the polymers was significantly affected by the molecular weight of the polymers. In general, increase in molecular weight of the polymer significantly increased the transfection efficiencies of polymers regardless of density of amine groups on the polymer surface, which was also accompanied by the higher toxicity of that polymer. In contrast, the transcription activity of genes into Xenopus oocytes showed lower levels of gene expression with the higher molecular weight of pTMAEM·Cl, which may be attributed to the higher stability of complexes and protection of DNA from polymerases or it could be due to the possible toxicological effects of higher molecular weight complexes in vitro. Thus, it can be concluded that the increase in molecular weight of the polymer can significantly improve their transfection efficiencies, but the incorporation of quaternary groups may lead to less efficient passage through the target cell to the nucleus [47]. Effect of Amine Numbers on the Complex Formation The effect of amine numbers of secondary amine-containing polymers on biophysical properties of polyplexes formed under physiological conditions was recently studied. For this purpose oligoethyleneamine-substituted trehalose polymers were synthesized with varying amine densities (Tr1-Tr4 containing 1–4 secondary amine/oligoethyleneamine monomers, respectively). Trehalose is a disaccharide, which is found to prevent protein adsorption on the gene delivery system. The hydrodynamic diameter of the polyplexes formed in buffer of pH = 7.4 was found to be independent of the polymer amine numbers at lower N/P ratios. At specific N/P ratios (ranging between

160

CATIONIC GLYCOPOLYMERS

1 and 2.5 for Tr1-Tr4) polyplex–polyplex aggregation was observed, due to the dominant van der Waals forces, upon the charge neutralization of complexes in physiological buffers [45]. However, the positive N/P ratios for secondary amine-based polymers exhibit complete condensation of DNA as shown by other amine-based polymers under the same conditions [48]. The library of secondary amine-based glycopolymers synthesized by Liu and Reineke were used to study the biological properties of polyplexes. It was demonstrated that transfection efficiencies of polyplexes is significantly affected by both the number of hydroxyl groups of glycopolymers and the number of amine units of the polymer. As, expected the increase in amine density, increased the gene expression level, regardless of the type of carbohydrate unit used in the glycopolymer segment. However, it was also shown that glucose based glycopolymers generally bind DNA at the lower N/P ratios, compared to galactose and mannose based glycopolymer, in the presence of same number of amine groups on the polymer segment, thus leading to better gene transfer ability. These differences in DNA binding ability of the cationic glycopolymers at the constant cationic ratios is attributed to the hydroxyl stereochemistry of the glycopolymers [28].

3.4 BIOLOGICAL APPLICATIONS The synthetic biomaterials can display multiple bioactivating elements that are of increasing interest for the study of cell-mediated interactions, drug delivery, and tissue engineering. Although the most studied function of cationic glycopolymers is their use as a gene delivery vehicle, which has been extensively explored and is the focus of this chapter, they can also be used for drug delivery purposes. For example, the major problem in ocular therapies is to provide and maintain adequate concentration of drug at the site of action. One of the most successful approaches in this direction is the use of colloidal suspensions such as polymeric nanospheres. The mucoadhesion of cationic polymers has been found to be useful for prolonging the precorneal residence time of the drug, predominantly due to the hydrogen bonding between the cationic polymers and the anionic mucin. For this purpose biodegradable polymeric nanocapsule-based drug delivery carriers were surface coated with natural cationic glycopolymer (chitosan). Chitosan is found to have interesting mucoadhesion properties due to the combination of electrostatic and hydrogen bonding interactions of the cationic glycopolymer. The PLL-functionalized nanocapsules were used as a control. The comparison of two types of drug delivery carriers was performed in vitro and in vivo, and it was found that chitosan-functionalized drug delivery carriers show enhanced activity and higher drug transfer capability both in vitro and in vivo, due to the significant hydrogen bonding capabilities and possibly due to the lack of agglomeration of nanoparticles in vivo due to carbohydrate component of the cationic glycopolymers, as discussed earlier [49]. The individual properties of glycopolymers and cationic polymers can also be utilized for their use in other biological and pharmacological applications [48]. The naturally occurring glycopolymers, usually taken as energy sources, are now known to

CONCLUSION AND FUTURE DIRECTIONS

161

have a variety of biological functions. For instance, a sulfated polysaccharide, heparin plays an essential role in blood coagulation, while hyaluronan like polysaccharides act as joint lubricant. Cell surface carbohydrates participate in various biological functions, including adhesion, recognition, and metastasis. These naturally occurring glycopolymers are heterogeneous and possess defined chemical structures. Synthetic carbohydrate-based polymers having pendant sugar residues are emerging as important tools and are of great interest in biochemistry and in medicines. They are found to have anticoagulant activity similar to heparin, can serve as vaccines, are involved in tissue engineering, and are used for the treatment of infectious diseases [33, 50–52]. For example, cell–cell and cell–surface interactions in the body are involved in a variety of pathological and physiological processes. Polyvalency, the ability of the ligand to bind to a target through multiple chemical interactions is a dominant factor in promoting the binding of pathogen to the cell surface. For example, influenza A, an orthomyxovirus responsible for the severe outbreaks of influenza adheres to the terminal N-acetyl neuraminic acid (Neu5Ac) of glycolipids and glycoproteins on the surface of host cells leading to hemagglutination. The synthesis of copolymers with Neu5Ac incorporated moiety has been used as a possible source to effectively inhibit the virus-induced agglutination of erythrocytes [33]. The cationic polymers can also be used to construct the sensors for DNA detection. The sensors based on conjugated polymers are found to be sensitive toward very minor perturbations due to the amplification by collective response, and thereof offer a remarkable advantage over small-molecule-based sensors. The combination of macromolecules (biological) with synthetic conjugated polymers can produce new hybrid biosensing elements as an interesting tool for biochemists, molecular biologists, and physicians. For example, polythiophene-based cationic molecules that exhibit interesting chromic features in the presence of external stimuli can be employed in biosensing apparatus, thus creating an assay that would not require any chemical manipulation or complex reactions. For example, at 55◦ C, an aqueous solution of cationic polythiophene is yellow with absorption wavelength near 400 nm. Upon the addition of one equivalent oligonucleotide, the duplex formation between polythiophene and oligonucleotides results in red color mixture with ␭max = 527 nm. Furthermore, the addition of one equivalent complementary oligonucleotide in the same mixture results in the restoration of yellow color, presumably due to the formation of triplex strand, obtained by the complexation of cationic polymer with hybridized DNA. These colorimetric effects are made possible due to the conformational changes in the morphology of cationic polymer, polymer in duplex form is planar and highly conjugated, while the triplex form of the polymer is nonplanar and slightly conjugated, displaying different colors at different morphologies [53].

3.5 CONCLUSION AND FUTURE DIRECTIONS The polymers and their derivatives are largely synthesized and utilized for various applications. The glycopolymers are unique for their possible applications for

162

CATIONIC GLYCOPOLYMERS

biological and biomedical applications. The advent of living radical polymerization has further eased the synthesis of glycopolymers and their corresponding copolymers, by providing the better control on the molecular weights and polydispersities of the synthetic polymer, in addition to bypassing the protected group chemistry steps. The glycopolymers and their copolymer produced are being used for gene and drug delivery as biosensors and for other biotechnology purposes. However, the synthetic glycopolymers prepared are based on simple monomer units and may not depict the true architecture of complex natural carbohydrate units. The need is to further explore the impact of using simple monomeric-unit-based polymers compared to complex natural polymers for biological applications. Moreover, more work is required to prepare complex glycopolymers to better mimic the natural conditions for their uses for biological applications.

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29. Liu, Y., and Reineke, T. M. (2006). Poly(Glycoamidoamine)s for Gene Delivery: Stability of Polyplexes and Efficacy with Cardiomyoblast Cells. Bioconjug. Chem. 17, 101– 108. 30. Ahn, C.-H., Chae, S. Y., Bae, Y. H., and Kim, S. W. (2004). Synthesis of Biodegradable Multi-block Copolymers of Poly(l-lysine) and Poly(Ethylene Glycol) as a Non-viral Gene Carrier. J. Control. Release 97, 567–574. 31. Brissault, B., Kichler, A., Guis, C., Leborgne, C., Danos, O., and Cheradame, H. (2003). Synthesis of Linear Polyethylenimine Derivatives for DNA Transfection. Bioconjug. Chem. 14, 581–587. 32. Deng, Z., Ahmed, M., and Narain, R. (2009). Novel Well-Defined Glycopolymers Synthesized via the Reversible Addition Fragmentation Chain Transfer Process in Aqueous Media. J. Poly. Sci. Part A: Polym. Chem. 47, 614–627. 33. Wang, Q., Dordick, S. J., and Linhardt, J. R. (2002). Synthesis and Application of Carbohydrate-Containing Polymers. Chem. Mater. 14, 3232–3244. 34. Labeau, M.-P., Cramail, H., and Deffieux, A. (1998). Amphiphilic Block Copolymers of Controlled Dimensions with Hydrophilic Glycosidic Vinyl Ether Moieties. Macromol. Chem. Phys. 199, 335–342. 35. Mortell, H. K., Gingras, M., and Kiessling, L. L. (1994). Synthesis of Cell Agglutination Inhibitors by Aqueous Ring-Opening Metathesis Polymerization. J. Am. Chem. Soc. 116, 12053–12054. 36. Descotes, G., Ramza, J., Basset, J.-M., and Pagano, S. (1994). Metathesis of -Unsaturated Glucosides with Chloro-aryloxide Complexes of Tungsten, as a New Way Leading to Unsaturated Bolaamphiphiles. Tetrahedron Lett. 35, 7379–7382. 37. Gotz, H., Harth, E., Schiller, M. S., Frank, W. C., Knoll, W., and Hawker, J. C. (2002). Synthesis of Lipo-Glycopolymer Amphiphiles by Nitroxide-Mediated Living Free-Radical Polymerization. J. Poly. Sci. Part A: Polym. Chem. 40, 3379–3391. 38. Chen, Y., and Wulff, G. (2001). Amphiphilic Block Copolymers with Pendent Sugar as Hydrophilic Segments and Their Surface Properties. Macromol. Chem. Phys. 202, 3273–3278. 39. Narumi, A., Matsuda, T., Kaga, H., Satoh, T., and Kakuchi, T. (2002). Synthesis of Amphiphilic Triblock Copolymer of Polystyrene and Poly(4-vinylbenzyl glucoside) via TEMPO-Mediated Living Radical Polymerization. Polymer 43, 4835–4840. 40. Lowe, B. A., and Wang, R. (2007). Synthesis of Controlled-Structure AB Diblock Copolymers of 3-O-Methacryloyl-1,2:3,4-di-O-isopropylidene-d-Galactopyranose and 2-(Dimethylamino)Ethyl Methacrylate. Polymer 48, 2221–2230. 41. Albertin, L., Stenzel, M., Barner-Kowollik, C., John, L., Foster, R., and Davis, T. P. (2004). Well-Defined Glycopolymers from RAFT Polymerization: Poly(methyl 6-OMethacryloyl-␣-d-Glucoside) and Its Block Copolymer with 2-Hydroxyethyl Methacrylate. Macromolecules 37, 7530–7537. 42. Narain, R., and Armes, P. S. (2002). Synthesis of Low Polydispersity, Controlled-Structure Sugar Methacrylate Polymers under Mild Conditions Without Protecting Group Chemistry. Chem. Commun. 2776–2777. 43. Deng, Z., Bouchekif, H., Babooram, K., Housni, A., Choytun, and N., Narain, R. (2008). Facile Synthesis of Controlled-Structure Primary Amine-Based Methacrylamide Polymers via the Reversible Addition-Fragmentation Chain Transfer Process. J. Poly. Sci. Part A 46, 4984–4996.

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CHAPTER 4

GLYCOPOLYMER BIOCONJUGATES MARYA AHMED and RAVIN NARAIN Department of Chemical and Materials Engineering and Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, Canada

4.1 Introduction 4.2 Bioconjugation Techniques 4.2.1 Random Bioconjugation 4.2.2 Site-Specific Bioconjugation 4.3 Applications 4.4 Conclusion and Future Directions References

167 170 171 172 181 186 186

4.1 INTRODUCTION The term bioconjugation is defined as the linking of two or more molecules to produce novel compounds in which the properties of the individual components remain unaltered [1]. The macromolecules of life, including carbohydrates, proteins, and nucleic acids, are complex ideal structures that have attracted researchers for decades, in contrast to synthetic polymers that are simple chemicals with diverse structures [2]. The bioconjugation of these natural and synthetic compounds can lead to the engineering of novel materials with the desired characteristics through a combination of their individual properties [1, 2]. These synthetic macromolecules produced by the bioconjugation approaches are termed polymer bioconjugates, biohybrids, or molecular chimeras [2]. The synthetic macromolecules such as polymers are far from nature due to their relatively simple structure; the motivation to produce polymer bioconjugates also comes from nature itself, where biological systems are found to be exploited at different levels [2, 3]. while the complex monomeric units of nature

Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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possess biological properties, the simpler monomeric units that are arranged into polymer chains produce self-organized structures. This natural phenomenon becomes apparent upon the study of carbohydrates, proteins, and nucleic acids at molecular level [2]. Thus, mimicking these basic rules of nature has lead to a field of research that first originated in biological sciences but is now found to have diverse applications [2, 3]. It is the versatility of bioconjugation techniques that has enabled their use in every discipline of life. For example, biological assays, diagnostics, and clinical testing are now based on bioconjugates [1, 4–8]. The field of polymer chemistry is rapidly advancing and a variety of polymers have been synthesized and utilized at industrial as well as academic levels. The bioconjugates of polymers have received enormous attention due to their extensive applications in medicines, biotechnology, and nanotechnology [1, 5, 6]. However, the choice of modification and conjugation techniques depends on the presence of reactive functional groups on the reagents and target molecules. The knowledge of the basic mechanism of conjugation and compatibility of reactive functional groups and the crosslinking reagents is thus necessary to yield the optimum results [1]. The major classes of macromolecules in nature include carbohydrates, nucleic acids, proteins, and lipids [8]. Proteins are the most common target for modification or conjugation purposes due to the presence of versatile functional groups present on their surface. Proteins are polymeric chains of amino acids, and it is the nature of amino acids that determine the functional and structural properties and reactivity of the proteins. Each amino acid is composed of an amino group and a carboxyl group bound to a central carbon, termed an ␣ carbon. The ␣ carbon also contains a side chain unique to each amino acid and a hydrogen atom linked to it [1]. The molecular structure of amino acid is shown in Figure 4.1. These side chains do not participate in peptide bond formation and are free to interact with the environment and to undergo other bioconjugation reactions. The protein molecules contain nine different types of alkyl groups that can readily participate in bioconjugation approaches. These nine amino acids are lysine, arginine, aspartic acid, glutamic acid, cysteine, histidine, tyrosine, methionine, and tryptophan, containing eight principal functional groups, including primary amines, carboxylates, disulfides or sulfhydryls, thioethers, imidazolyl, guanidinyl groups, and phenolic and indolyl rings. The nucleophilicity of these major groups in biological molecules provides an explanation to the trend in their reactivity [1, 9]: R S > R NH2 > R COO− = R O− The polymer protein conjugate is a dominant field in chemistry and is found to have various applications in biotechnology, pharmaceuticals, and life sciences H COOH

R NH2

FIGURE 4.1 Chemical structure of basic amino acid. (Adapted from Ref. [1].)

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[5, 6, 8, 10–13]. One of the aspects to produce polymer protein conjugates, in addition to imparting the functionality and mimicking the naturally occurring macromolecules, is to provide the protection and solubility to the targeted proteins, thus making them water soluble and resistant to proteolytic degradation. The first polymer–protein bioconjugate was prepared by the conjugation of polyethyleneglycol (PEG) to the protein at lysine residues. Several polymer–protein bioconjugates have been synthesized and are shown to possess various biological properties. These polymer conjugates are, however, largely limited by the methodologies available for their synthesis due to the limitation in the number of conjugated drugs/dyes and due to their cytotoxicity [11]. A novel approach to solve these problems is bioconjugation of proteins with carbohydrate-based polymers. The glycopolymers themselves have great potential as gene and drug delivery vehicles, tissue culture substrates, and for macromolecular assemblies. The conjugation of glycopolymers with macromolecules is thought to enhance the functions of conjugates, and they can now be used to study various biological phenomena at molecular levels, in addition to serving their native functions [3]. The synthesis of glycopolymer protein bioconjugates is relatively new and is a rapidly emerging field of polymer chemistry due to the wide range of applications of these neoglycoproteins in various fields of science [14]. The ideal glycopolymers used for bioconjugation purpose should contain functional groups for their conjugation to proteins and other molecules, in addition to imparting solubility and biocompatibility to the system. The carbohydrates are interesting due to the variations in their structures, complexities, and conjugation to other macromolecules in nature. The carbohydrate moieties attached to other macromolecules are termed oligosaccharides [15]. No single unified function of oligosaccharides has yet been identified; instead, their major function is to serve as the recognition marker and to modify the intrinsic properties of macromolecules, such as the proteins to which they are attached. Glycosylation is highly sensitive to alteration in cellular functions, thus the control of glycosylation by the cells is usually achieved by the use of recognition markers on the surface of biomolecules to avoid copying this information into the DNA (deoxyribonucleic acid) of that protein [15]. In aqueous solutions, monomeric units of carbohydrates predominantly exist in cyclic form due to the tendency of aldehyde or ketone groups to undergo intramolecular cyclization with its own OH groups to produce hemiacetal and hemiketal structures. The conjugation reactions to monomeric units of carbohydrates are thus designed to target these limited functional groups present on the surface of simple carbohydrates [15, 16]. The synthesis of glycopolymers by various polymerization techniques incorporates various functional groups at glycopolymer ends, enabling their facile conjugation to other macromolecules [4–10, 17]. In addition, the glycopolymer-based carbohydrate protein interactions are multivalent in nature and differ from those of monovalent interactions. These multivalent interactions are found to play a crucial role in biomolecular recognition events. The synthesis of glycopolymers of controlled dimensions is challenging and various polymerization techniques have been used for this purpose. The examples include cationic polymerization, ring-opening polymerization, ringopening metathesis polymerization (ROMP), nitric-oxide-mediated polymerization

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FIGURE 4.2 Telechelic glycopolymers produced by LRP containing a variety of functionalized groups. (Reproduced with permission from Ref. [9].)

(NOMP), and living radical polymerization (LRP) [9, 18]. However, the synthesis of glycopolymers of increased complexity and controlled molecular weight usually involves several steps of protection and deprotection, and polymers are further modified by postmodification approaches to produce end-functionalized polymers for bioconjugation purposes [16]. A versatile approach to produce polymer–protein bioconjugates stems from the successful synthesis of well-defined glycopolymers by LRP. This approach has allowed researchers to synthesize well-defined glycopolymer–protein conjugates in the absence of protecting group chemistry. One of the most widely used ligands in this context is biotin. Biotin-functionalized glycopolymers have been largely prepared and are further employed for the conjugation with sterptavidin [19]. With the advent of free radical polymerization, it was possible to produce glycopolymers of controlled molecular weight in the absence of protected group chemistry in aqueous solutions. In addition, the technique was tolerant to a wide range of functional groups, producing telechelic glycopolymers for bioconjugation purposes. These groups include OH, NH2 , COOH, SO3 , OCN, and biotin. The schematics of these end-functionalized polymers by LRP are shown in Figure 4.2 [9]. The advent of living radical polymerization has enabled the synthesis of welldefined glycopolymers with end-group functionalities, owing to highly tolerant properties of the technique toward different types of monomers, solvents, temperature, and functional groups [17]. The protein–carbohydrate interactions are prevalent in nature and will be the focus of this chapter; however, bioconjugation of peptides and nucleic acid with glycopolymers will also be discussed.

4.2 BIOCONJUGATION TECHNIQUES Two major approaches to produce novel bioconjugates include postmodification of functionalized polymers to biomolecules and the in situ polymerization of monomers directly at the site on the biomolecules [9]. Both techniques have their own advantages and drawbacks. For example, the postpolymerization conjugation approach requires synthetic polymers with terminal functional groups, which can be introduced either during the polymerization process or by postpolymerization modification, and the process is followed by extensive purification steps resulting in low yield of the final product. The in situ polymerization approaches utilize biomolecules modified

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171

with polymerization controlling agent (i.e., initiator, chain transfer agent) and seem advantageous compared to postpolymerization technique with respect to purification, yield, specificity, synthesis, and controllability. However, polymerization conditions may affect the sensitivity of the biomolecules used in the process [9, 18]. These two major bioconjugation approaches are reported to produce novel glycoconjugates, which may be a modified site specifically or randomly with the molecule of interest. This random or site-specific modification of glycopolymers is based on the presence of reactive end-functionalities on both glycopolymers and macromolecules [18]. 4.2.1 Random Bioconjugation Protein–carbohydrate multivalent interactions are dominant in nature and make the first site for the attachment of pathogen to the host surface. To better understand these multivalent interactions, glycopeptides are synthesized by using bioconjugation approaches and are then used to detect bacterial toxins. The bioconjugation of aminefunctionalized glycomonomers with COOH (glutamic acid) containing polypeptides is shown by postmodification approach in Scheme 4.1. The activation of glutamic acid residues of polypeptides was obtained using 2(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) resulting in amide bond formation with N(ε-amino caproyl)-␤-d-galactopyranoside under basic conditions. The excess of carporyl-functionalized galactopyranoside and small molecules were removed by dialysis and the purity of the samples was detected using high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Although the process of bioconjugation is random, all glutamic acid molecules present on the surface of polypeptide are modified with glycomonomers

OH O DNA +

HOOC

HO NH HO

NH2

HO O

DMSO HBTU/DIEA OH O

O

HO

NH HO

N H

HO

DNA

O

SCHEME 4.1 Postmodification of ␣-helical oligopeptide by N-(ε-amino caproyl)-␤-dgalactopyranoside. (Reproduced with permission from Ref. [20].)

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under this condition. The approach is suitable to provide the multivalent affect of carbohydrate monomers to detect the bacterial toxin. Furthermore, the crystal structure of shigalike toxins reveals the presence of two binding sites for glycomonomers ˚ distance; thus, design of this scaffold is found to on the surface of toxin at ∼35 A enhance the sensitivity of glycopeptides toward the bacterial toxin [6, 20]. The synthesis of polymers with varying functional groups is a rapidly advancing approach in polymer chemistry and is also responsible for dictating the applications of these polymers. Polymers bearing aldehyde groups are versatile due to their applicability in biomedicines, and as aldehydes they are reactive under mild conditions and can form stable covalent bonds with hydrazine and amino groups, resulting in Schiff bases and hydrazone linkages, respectively. These aldehydefunctionalized polymers can thus be used for enzymes, drugs, and protein modification purposes. Xiao et al. has used this strategy to produce glycopolymer–protein bioconjugates of controlled molecular weights [21]. A novel galactose-based monomer containing an aldehyde group, namely, 1,2:3,4-di-O-isopropylidene-6-O-2 -formyl4 -vinylphenyl)-d-galactopyranose (IVDG), was synthesized and polymerized by reversible addition–fragmentation chain transfer (RAFT), as a first step toward glycoprotein synthesis. The resulting glycopolymers obtained were found to be amphiphilic in nature and were used for bioconjugation purposes. Bovine serum albumin (BSA) protein is the most abundant protein in nature and is commercially available; the conjugation of BSA to glycopolymers was obtained by Schiff base formation. The immobilization of BSA on the surface of micelles was simply performed by mixing the two macromolecules in aqueous solution for 8 h to produce Schiff base linkage [21, 22]. The resulting protein-immobilized poly-IVDG micelles were further characterized using dynamic light scattering (DLS) and transmission electron microscope (TEM) to confirm the presence of protein on the surface of micelles [22]. 4.2.2 Site-Specific Bioconjugation One of the aims of conjugation approaches is to create a stable product with the retention of activity and native state of macromolecules. One way to achieve this goal is by avoiding the chemical reaction at the active center of the biomolecules, which can be effectively done either by protecting the active site or by choosing a functional group away from the active site for conjugation purposes. The active center can be defined as the portion of the macromolecules, for example, proteins that specifically interact with other substances, as a part of its physiological function. The protection of the active site of the protein involves complex procedures and may cause irreversible damage to the macromolecule. The site-directed conjugation away from the active site is another suitable solution to this approach. The presence of these specific functional groups away from the active site on the three-dimensional structure of macromolecules provides the appropriate source for site-specific bioconjugation by postmodification approach [1]. In addition to targeting the limited number of functional moieties on the surface of targeting molecules, researchers have implied various polymerization techniques to achieve the site-specific bioconjugation of glycopolymers with molecules of interest.

BIOCONJUGATION TECHNIQUES

X

Incorporation

Amino acid initiator

173

ATRP X

Monomer

Peptide macroinitiator

SCHEME 4.2 Synthesis of serine-based ATRP initiator to perform in situ polymerization of O-acetyl-d-glucosamine (O-GlcNAc). (Reproduced with permission from Ref. [22].)

The facile synthesis of glycopeptides by in situ polymerization has been demonstrated by Broyer et al. [22]. In this approach, the glycopolymer is grown from a predetermined amino acid chain of the peptide in an effort to produce site selectively modified peptides with glycopolymers [22] (Scheme 4.2). The in situ polymerization requires the synthesis of macromolecule-based initiators, which can then be used to conjugate the biomolecules of interest site specifically. For this purpose, serine-based unnatural amino acid containing atom transfer radical polymerization (ATRP) initiator (2-bromo isobutyrate) was first synthesized and was then incorporated into the amino acid chain. O-Acetyl-d-glucosamine (O-GlcNAc). These steps are shown in Scheme 4.2. O-GlcNAc is a well-studied posttranslational modification that occurs naturally on various proteins either at serine or threonine residues, and the errors in this postmodification approach leads to various fatal diseases [17]. In an effort to mimic the physiological conditions and to better understand the impact of these glycosylation steps in vivo, O-GlcNAc-functionalized glycopeptides have been prepared by various polymerization techniques. The peptide chosen for this study was VMSVVQTK, as it is found to be a naturally modified O-GlcNAc peptide in human cellular factor protein, thus making it an excellent model for site-specific modification purposes. O-GlcNAc polymer-modified peptides were produced by ATRP using serine-based initiator discussed above. The glycopeptides of 15 kDa with low polydispersity (1.19) thus produced were detected by gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1 H-NMR) [22]. The ratio of thiol groups on the surface of protein is very small and can be ideally targeted for site-specific conjugation purposes. One example of the synthesis of neoglycoproteins by site-specific postmodification approach is shown in Scheme 4.3. The first step in the synthesis of neoglycoprotein was the synthesis of end-functionalized glycopolymers. The synthesis of N-acetyl-d-glucosamine (GlcNAc)-based glycopolymer containing pyridyl disulfide end groups was obtained by Dorbatt et al. [17] and was used to polymerize glycomonomers via ATRP. The well-defined glycopolymers thus obtained were dialyzed to remove the impurities, and glycopolymers of Mn 10.2 kDa were thus achieved with narrow polydispersity (Mw /Mn = 1.12). The bioconjugation of these glycopolymers with macromolecules was then achieved site specifically by postmodification of glycopolymers with macromolecules, including proteins and nucleic acids. GlcNAc-based glycopolymers

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H N

Val-Met

OH

O Val-Val-Gln(Trt)-Thr(tBu)-Lys(Mca)-Gly-OH

O

O

HO HO

O

O

NHAc

O Br

CuBr, bipy, MeOH:H2O; 4:1

O

H N

Val-Met

O Val-Val-Gln(Trt)-Thr(tBu)-Lys(Mca)-Gly-OH O O

HN

Lys(Mca) R =

n O

O

O O

OH O

HO HO

O

O O

NHAc

SCHEME 4.3 Synthesis of O-acetyl-d-glucosamine (O-GlcNAc)-modified polypeptides by ATRP. (Reproduced with permission from Ref. [22].)

produced were conjugated to the protein at the thiol end by disulfide bond formation as shown in Scheme 4.4 [9]. The obvious advantage of these neoglycopolymer–protein complexes is to provide the solubility and protection to the biomolecule attached in addition to serving as a targeting moiety. The versatility and bioconjugation ability of these glycopolymers were also then demonstrated by their conjugation to short interfering ribonucleic acid (siRNA) via disulfide bond exchange. siRNA is a double-stranded RNA molecule that can be synthesized chemically using desired end-group functionality. siRNA with free thiol groups is synthesized, and their conjugation efficiency with pyridyl-disulfidefunctionalized glycopolymers is assessed using agarose gel electrophoresis assay, followed by staining with nucleic-acid-sensitive fluorescent dyes. The quantification of the image intensity of free siRNA compared to polymer-conjugated siRNA revealed 97% conjugation efficiency. It was also demonstrated that the process of

SH

O N

S

S

n O 10 mM NaHCO3, 21°C, 24 h

O

HO HO

O S

S

n O

O

O

O

OH O O NHAc

OH O O OH OH

NHAc

SCHEME 4.4 Site-specific conjugation of BSA to pyridyl disulfide end groups functionalized N-acetyl-d-glucosamine (GlcNAc)-based glycopolymers. (Reproduced with permission from Ref. [17].)

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175

conjugation is reversible and the treatment of glycopolymer–siRNA complexes with dithiothreitol (DTT) leads to the release of siRNA from the polymers, suggesting the suitability of this polymer for gene and drug delivery purposes [17]. Although the nucleic acids–glycopolymer conjugates exist in nature, the conjugation of these polymers to higher order macromolecules such as nucleic acids is less explored due to the complexity, denaturing ability, and degradability of nucleic acid molecules [2]. Glycosylated bases in DNA were first discovered in viruses. For example, DNA of T-even phages contains 5-hydroxymethyl cytosine (instead of cytosine) in which DNA is glycosylated via the O-glycoside bond. In addition rabbit and rat livers are shown to contain RNA-bearing glycosylated bases. These glycosylated structures are thought to protect the DNA/RNA from degradation. However, the precise function of this modification can only be detected if glycoconjugates of DNA of controlled architecture can be synthesized, without affecting the native structure of DNA. These artificial glycoconjugate materials can also be useful for the purpose of cell-targeted gene therapy, due to the cluster glycoside effect produced by the multivalent interaction of glycoconjugates with proteins present on the cell surface. However, facile introduction of multiple carbohydrates on a single DNA chain is very challenging. Although other strategies including solid-phase synthesis and chemical modification of DNA molecules have been used to introduce saccharide moieties on the surface of nucleic acids, these strategies are limited by multiple tedious steps and the introduction of single sugar moiety to each DNA molecule, respectively. The complex formation between DNA and saccharide due to hydrogen bonding has also been investigated. However, these complexes are found to dissociate upon higher dilutions. The diazo-coupling method is one approach to introduce multiple oligosaccharide chains on a single DNA molecule. This modification is done at position 8 of the guanine base present at the major groove of the DNA molecule and thus is found to minimally interrupt the hydrogen bonding between DNA duplexes. The glycoconjugates of DNA were prepared by first synthesizing benzene diazonium derivatives containing lactose and cellobiose moieties, which were further conjugated to DNA via diazo-coupling. This process involves multiple steps of protecting and deprotecting of saccharide molecules to yield diazonium salts that are then conjugated to DNA molecules. The salmon testes DNA was sonicated and allowed to react with excess diazo compounds in borate buffer at 25◦ C. The bioconjugate complexes obtained were extensively purified by precipitation and ion exchange column chromatography to yield pure yellow fibers. The glycopolymer content of the conjugates were determined using enzyme-based colorimetric assays and further characterization of conjugates was done using PAGE and circular dichroism (CD) spectra [23]. Not all macromolecules have naturally occurring free site-specific functional groups such as cysteine on the site of interest. In this case, the specific functional groups can be introduced at the selected site by other chemical approaches [9, 16, 24]. A reaction that has proven useful in this regard is the Sharpless-modified Huisgen cyclization of alkyne and azide [24]. Although the Cu(I) conditions required for the reaction leads to cell death, this reaction has been used successfully for the modification of viruses and proteins on the cell surface. For this technique, the first step is the surface modification of biomolecules with unnatural functional groups including

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iodo, keto, and alkenyl groups [25]. The clickable polymers is a promising approach to modify neoglycopolymers site specifically by the postfunctionalization approach. For example, mannopyranoside containing fluorescent polymers were synthesized by the clickable approach and their interaction with mannose binding lectin (MBL) was explored by Geng et al. [26]. MBL is a mammalian lectin of the immune system, and mannoside structures are found to be prevalent on the surface of pathogens including bacteria and fungi, but these structures are distinct from mannose of human cells. MBL binding to mannosides triggers the cascade pathway of the immune system, which leads to opsonization and phagocytosis of pathogen and cell destruction. In this study BSA, a commercially available 66-kDa protein, is used to synthesize neoglycoproteins by various bioconjugation approaches, which are then used to study MBL-based interaction for further biological studies. Mannose-based polymers are also interesting as many mammalian lectins are found to interact with mannose-containing structures on the surface of pathogens. Maleimide end-functionalized polymers were first prepared by both postpolymerization strategy and by in situ polymerization of mannosebased monomers by ATRP and were then conjugated to BSA by using maleimide chemistry. Both techniques lead to the production of telechelic fluorescent glycopolymers of controlled dimensions. During in situ polymerization, mannose-containing monomers were first prepared by Huisgen 1,3-dipolar cycloaddition of the mannose azide to propargyl methacrylate using (PPh3)CuBr as a catalyst. The monomer was then polymerized using a maleimide-protected initiator along with fluorescent rhodamine B comonomer, using iminopyridine/Cu(I)Br system. (Scheme 4.5) [26]. In the second strategy propargyl-methacrylate-based polymers were prepared as a scaffold to react efficiently with azide-containing mannose. The polymerization of propargyl methacrylate was obtained using the Cu(I)Br/iminopyridine system in the

OH

O + HO HO

O

OH

OH O

a N3

(1)

HO HO

O

O

O OH O

N N–N

O

b

O

O HO

O

(2)

O

N

O

HO

HO HO

(a)

O

n O

O

Br m O

(3) N N–N

O

R c

O O O

R O O

N

O

O

N

Br O

(8) O

N

N

HO

Cr N

O

O

N

O

O HO

O

n O

O

Br m O

(4) O

HO HO

(9)

O

N N–N

R

O O

O d O Si

O

O

O

(5)

(b)

f

O O

N

O

n O O

(6)

Br m O

e

O

N O

O O

O

n O

O

Br m O

(7) Si

R

R

SCHEME 4.5 Synthesis of mannose-based fluorescent glycopolymers by (a) in situ polymerization and by (b) post polymerization. (Reproduced with permission from Ref. [26].)

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O BSA

BSA

SH PBS pH 7.0 DMSO

O

N

(4a) S HO HO HO

O

O HO

O

O

n O

O

N N N

Br m O N

N

O

O

(10) N

O

Cl− N+

SCHEME 4.6 Bioconjugation of cys 34 of BSA to maleimide-functionalized glycopolymers. (Reproduced with permission from Ref. [26].)

presence of initiator using protected group chemistry. The polymers obtained were then deprotected and were further conjugated to azide-containing glycomonomers by the click approach. The maleimide-terminated polymers thus obtained were then studied for their conjugation with BSA to yield glycoprotein mimetics using maleimide chemistry [26] (Scheme 4.6). Bovine serum albumin can be site specifically conjugated to maleimide-containing polymers due to the presence of one free thiol at the cysteine 34 position of the protein. Once the glycoprotein mimetics were obtained, their recognition by lectin was studied. The lectin receptor recognition was found to be dependent upon the length of polydentate ligands as well as the density of the binding epitope present in the neoglycopolymers. The binding studies of BSA–glycopolymers with MBL of rat were carried out using surface plasmon resonance to detect BSA–bioconjugate interactions. The unmodified BSA was used as a control. The conjugated and unconjugated BSA were immobilized on the surface of sensor chips, and sensograms were obtained in the presence of MBL in fluid phase. The results demonstrated the clear and dosedependent MBL binding to glycoconjugates, in the physiological range in contrast to BSA alone where no binding was observed. Further functional analysis was performed using enzyme-linked immunosorbent assay (ELISA) that showed that bioconjugates are able to activate the complement system via lectin pathway. This is an example where, by site-directed modification, biological molecules with dual characteristics were obtained. Protein components can potentially offer functions such as ligand binding, enzymatic activity, and antigen presentation in the case of immunological studies, whereas glycopolymers confer selective lectin binding and attachment to other cells and tissues. In short glycoproteins are potent biological molecules that are active due to the combined function of both protein and carbohydrate components. Moreover, these biohybrid structures were found to possess enzymatic activity of BSA; thus, it was confirmed that chemical modification did not affect the innate function of conjugated protein [26]. The detailed study of biological activity of glycans requires their incorporation in living cells. Neoglycoprotein complexes prepared by bioconjugation techniques

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GLYCOPOLYMER BIOCONJUGATES

can help understand the detailed analysis of these biological activities, if these neoglycoproteins are incorporated in the cell surface. The heterogeneous nature of cell membrane, however, challenges the modification of living cells. One way to solve this problem is genetic engineering, but this approach is found to be least effective for carbohydrates and lipids modifications. In addition, synthesis of artificial membranes and their modification with glycan structure is limited by the absence of the biological phenomenon occurring in these membranes. The common and major drawback to both of these above-mentioned approaches is their reliance on cellular machinery. An alternative approach is the passive insertion of well-defined glycan structures in the membrane of cells. This technique includes the modification of carbohydrates with hydrophobic groups such as fatty acids and their incorporation on the cell surface. The glycolipids and glycophophatidyl-anchored proteins are examples of this technique. Mucins are a class of glycoproteins that are rich in serine or threonine residues bearing O-linked glycans that play an important role in modulating cellular interactions. The altered functions of mucins are associated with many diseases; however, details of these mucin-dependent molecular interactions are not known. These mucin mimics can be artificially produced by synthesizing glycopolymers with lipophilic backbone for their facile insertion in cell membrane. For example, Rabuka et al. synthesized mucin mimics by incorporating synthetic glycans on the surface of polymethyl vinyl ketone (pMVK) by oxime linkage and by further incorporating a hydrophobic moiety such as pyrene, single chain lipids, and phospholipids at the end of the polymer chain to ensure their insertion in cell membrane. The first step toward the synthesis of mucin mimics was the synthesis of hydrophobic end-functionalized pMVK by RAFT. The lipophilic polymers thus obtained were modified by glycans and oxime linkage during the postmodification process. In addition a fluorescent probe (Texas red) was attached to the ketone group of the polymer using hydrazide linkage. These fluorescently labeled glycopolymers were used to determine the amount of polymer incorporated into the cell membrane. It was also found that incorporation of glycans into cell membrane is independent of the type of glycans attached to the polymer but is dependent on the presence of hydrophobic moiety of the polymer [27]. Self-organization strategy is widely used by the biomolecules in nature. For example, in cartilage, various glucosaminoglycans (GAGs) are attached to the protein core resulting in aggrecan structures that can then noncovalently interact with the hyaluronan backbone. These self-organization strategies are now increasingly appreciated and have been employed to synthesize nanomaterials with diverse biological properties. Antisense oligonucleotides (ODNs) are promising construction material that serves as a “molecular glue” to construct the macromolecules with controlled dimensions. The size and stability of DNA-modified macromolecules can be controlled by using ODNs of varying lengths and by changing the temperature, solvent, and pH of the external medium. Due to these attributes, ODNs are useful tools to synthesize glycoclusters using self-organization approach as shown in Scheme 4.7 [28]. A facile method to produce glycopolymer-grafted ODNs was introduced by Akasaka et al. [28]. Amine-functionalized glycopolymers were first modified by Niodoacetoxy succinimide in 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and the functionalization of polymers was quantified by using a ninhydrin reagent. The

BIOCONJUGATION TECHNIQUES

179

SCHEME 4.7 Synthesis of ODN–glycopolymer conjugates by self-organization strategy. (Reproduced with permission from Ref. [28].)

block-type ODN–glycopolymer conjugates were then synthesized by the conjugation of iodoacetamidated glycopolymers with thiol containing commercially available ODNs in the presence of triethylamine, as shown in Scheme 4.8. The self-organization of block-type ODNs with complementary ODNs produced graft-type assembly. These novel neoglycoconjugates produced can be utilized to study the interactions of lectins with glycoclusters [28]. In addition to covalent linkages, noncovalent interactions are found to play a dominant role in nature and are hence useful to establish various bioconjugation approaches for the facile coupling of biomolecules. The carbohydrate–protein and protein–protein interactions are important examples of this phenomenon. The glycopolymers that bind to the ligand-binding sites are interesting for the bioconjugation

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GLYCOPOLYMER BIOCONJUGATES

R1 R2 AcO

OAc R2 O

R1

c O

R1

X

1 2 β-Gal: R = OAc, R = H

b

α-Man: R1 = H, R2 = OAc

3 : X = NH2

d

R2 HO

R1

a

2 : X = N3

1

O N H

O 4

1 : X = Br

R

R2 AcO

OAc 2 R O

H

OH 2 R O

O O

R1

e

N H

5

OH 2 R R O 2 R HO R1

S

NH2

O NH

1

O n

Poly(Gal or Man)n-NH2 β-Gal : R1 = OH, R2 = H 2 α-Man : R1 = H, R = OH

Reagents and conditions: (a) NaN3, DMF, 65°C, 1 h, (b) H2 gas, Pd/C, THF, rt, 40 min, (c) methacryloyl chloride, triethylamine, THF, rt, 30 min, (d ) sodium methoxide, methanol, rt, 6 h, and (e) cysteamine, AAPD, water, 60°C.

SCHEME 4.8 Synthesis of block-type galactosylated ODNs. (Reproduced with permission from Ref. [28].)

purposes and can be easily synthesized by LRP techniques, containing proteins as the functional end moiety [7, 19]. Biotin is a naturally occurring ligand for avidin and streptavidin [19]. The biotinylated polymers can easily be synthesized by using biotinylated initiator during ATRP to produce biotinylated glycopolymers, which are ideal to produce self-organized polymer–protein hybrids due to molecular recognition between biotin and streptavidin (1013 –1015 M−1 ). The first step in this bioconjugation process is the synthesis of biotin-conjugated initiator, which is then utilized to polymerize protected GlcNAc monomers, as shown in Scheme 4.9. The neoglycopolymers produced were deprotected and were tested for the conjugation ability of biotinylated polymers to streptavidin. The biological interactions involved in biotinylated GlcNAc-glycopolymers with streptavidin-coated surfaces were used to study surface plasmon response (SPR). SPR is a powerful technique to observe the binding events [7]. Similarly, biotin end-functionalized glycopolymers were prepared by Sun et al. using cyanoxyl-mediated free radical polymerization. The arylaminefunctionalized biotin was used as an initiator to produce telechelic polymers and their biotinylated activity was detected using SPR response [29].

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APPLICATIONS

O HN H

NH H

O

3 H N

S

O

Br

O

Biotin

CuBr, Me6-TREN, DMSO

O

O

N H

O

O

O

6

5

n

AcOAcO

O OAc O

O

NHAc

4 CuBr, Me6-TREN, DMSO

O Biotin

N H

O

O

NaOMe

n O O

CHCl3/MeOH

OH O

HO HO

O

NHAc

7

SCHEME 4.9 Synthesis of biotinylated glycopolymers by ATRP. (Reproduced with permission from Ref. [7].)

An alternative method to detect biotin–streptavidin interactions is by using HABA–streptavidin assay. 4 -hydroxyazobenzene-2-carboxylic acid (HABA) interacts with streptavidin, and this interaction is indicated by a color change from yellow to red, which is indicated by a red shift in absorbance spectroscopy (␭ = 350 to ␭ = 500 nm). Isocyanate end-functionalized (OCN) end-functionalized polymers also provide a facile platform to conjugate glycopolymers with other amine-containing biomolecules. These cyanate end-group-functionalized polymers can also be converted into OH group-terminated glycopolymers by their treatment with water and pyridine [9]. This modification further enables their selective conjugation to the variety of amine-containing macromolecules [10, 19]. 4.3 APPLICATIONS The glycopolymer bioconjugates are found to have numerous applications in biomedicines, bionanotechnology, pharmaceuticals, and gene delivery. A few examples will be discussed here. The development of anti-influenza drugs is one of the major outcomes of bioconjugation approaches. The sialyl-glycopolymers can be enzymetically synthesized and chemically linked to the chitosan backbone. The glycopolymers produced were then used to detect the virus inhibition activity on Madine-Darby canine kidney cells. The inhibition of virus activity was almost 100% when cells were treated with sialyl-glycopolymer-linked chitosan. This inhibition of activity was based on the interaction of virus envelope proteins (hemagglutinin, HA) to sialic acid residues present on the surface of the glycopolymer. It was found that monomeric sialic acid units were unable to provide this inhibitory effect; however, multivalent interactions of sialic acid residues present on the surface of glycopolymers with HA produced the cluster glycoside effect, thus enhancing the inhibition activity [4, 30].

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GLYCOPOLYMER BIOCONJUGATES

Fluorescent-labeled glycopolymers are attractive for biosensing applications as they combine the scaffolding and imaging properties in one platform. Mannosemodified polymers are found to serve as a conjugation scaffold for Escherichia coli. Type I pilli in E. coli contain the FimH protein, which selectively binds with d-mannose residues. The incubation of fluorescently labeled mannose polymers with FimH containing a strain of E. coli results in the formation of fluorescently labeled bacteria from which polymer could not be removed even after washing. These fluorescent glycopolymers serve as a simple detection method in recognizing and detecting certain strains of bacteria [13]. In addition, similar strategy was implied to enhance the bacterial detection by externally controlled parameters, including the synthesis of thermoresponsive glycopolymers [12]. Detection of biological toxins is another important aspect for human survival. The biochemical approaches such as ELISA, polymerase chain reaction (PCR), and mass spectroscopy have been used for this purpose. An alternative method for the detection of biological toxins is a glycotechnology-based approach, which is based on the infection process itself. Ricin is a toxic compound with a median lethal dose (LD50 ) of 3–5 ␮g/kg for humans, which is much higher than hydrogen cyanide. The increased awareness of chemical and biological terrorism requires rapid detection of this toxin. Ricin consists of two B chains containing two ␤-lactoside binding domains. The rapid detection of the toxin is established by synthesizing and immobilizing lacto-lipids on the surface of the detector chip, as shown in Figure 4.3. Therefore, polyacrylamide-based glycopolymers were first prepared and were then immobilized on the gold sensor chip for ricin recognition by SPR. The competitive binding assays revealed that lactose-based glycopolymers inhibited the adsorption of ricin toxin dose dependently, and the inhibition of toxin was found to be the function of sugar density on the surface of polymers. The glycopolymers with higher lactose density showed

FIGURE 4.3 Detection of ricin toxin using bioconjugation approach. (Reproduced with permission from Ref. [31].)

APPLICATIONS

183

higher activity and vice versa, suggesting the usage of these glycopolymers in clinical applications [31]. Organic polymers are interesting due to their applications in various optical and electronic fields. Polythiophene (PT) is an important organic polymer that possess planar to nonplanar conformational transitions of the ␲-conjugated system, and these materials show excellent optical properties upon excitation by heat, metals, chemicals, and proteins. These chromic transitions of materials are used for toxin detection purposes; however, for their applications in biotechnology these polymers must be tailored for biological systems. Carbohydrates are small molecules found external to the cell surface and are involved in a variety of key events. They are more desirable than proteins in imparting the biocompatibility to the foreign materials due to their smaller size. Thus biocompatible thiophene-based materials can be produced upon their functionalization with carbohydrates. Sialic acid and mannose-based thiophene copolymers were synthesized and their bioconjugation abilities were used to detect different bacterial toxins using optical assays. To demonstrate the biochromic capabilities of thiophene glycopolymers mannose-PT and sialic-PT were incubated with Concanavalin A (Con A) and Wheat Germ A, respectively. The toxin detection appeared as a red shift in ultraviolet (UV) absorbance, as discussed above [5]. The interaction of selectins with glycosylated proteins triggers a cascade of events leading to inflammatory and cell-mediated immune response. The glycosylated proteins involved in this phenomenon are a sulfated analog of tetrasaccharide sialyl Lewis x, which are potent inhibitor of selectins. However, their synthesis in the lab poses various problems, including weak affinity, short circulation time in blood, and exhaustive synthetic routes. The relatively simple therapeutic analogs for selectin binding can be prepared by bioconjugation of oligosaccharides with branched polymers. Rele et al. synthesized well-controlled branched polyethyleneoxide (PEO) polymers that served as a scaffold for selectin binding. The hydroxyl-terminated PEO were conjugated with trichloroacetamide ␤-lactose octaacetate resulting in covalent attachment of acetyl-protected lactose residues on PEO dendrimers [32]. The glycopolymers obtained were deprotected, followed by lactose sulfation. The final products were then used to limit the inflammatory responses in vivo by using selectindependent blockade. The inflammation response induced in mice was found to be reversible upon treatment with PEO-based glycopolymers (Fig. 4.4). Heparin is a competitive natural inhibitor of selectin and is found to greatly reduce the immune response. However, a major drawback of this macromolecule is its anticoagulation ability, which limits its use in clinical trials. The PEO-based glycopolymers synthesized are found to have effects comparable to heparin in the absence of abovementioned drawbacks [32]. The carbohydrate–protein bioconjugation techniques are now widely employed in the field of research to further explore the biological events in complex systems of eukaryotes. The glycopolymer modified microparticles have been synthesized to study the mechanism of pathogen recognition by antigen presenting cells (APCs) based upon carbohydrate-protein interactions. Although the pathogen recognition is extensively studied, it is hard to interpret if the process occurs due to ligand–receptor interactions or due to the presence of more than one signal recognition event. To

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GLYCOPOLYMER BIOCONJUGATES

FIGURE 4.4 Schematics of interactions of selectins with galactosylated polymers. (Reproduced with permission from Ref. [32].)

answer these questions, mannan-based glycopolymers were synthesized and were immobilized on the surface of microparticles to achieve their three-dimensional configuration. Mannan is a major antigen of yeast cells and serves as a multivalent ligand for toll-like receptors (TLR). It is a polysaccharide consisting of ␣-(1,6)-linked mannose backbone grafted with ␣-(1,2) and ␣-(1,3)-linked mannose side chains, some of which carry phosphate groups, which serve as a natural ligand for the receptors of APCs. It was hypothesized that mannoside ligands linked to the surface of microspheres at high densities can serve as a strategy to mimic high-molecularweight mannose-based pathogen-associated molecular patterns (PAMPs) with synthetic small molecular ligands. The results obtained based upon the bioconjugation of mannan-functionalized spheres and receptors of APCs indicated that mannoside density was the first factor responsible for the phagocytosis of microspheres. It was also found that the recognition of the mannoside-enriched surface alone cannot serve as a trigger for opsonization; thus, more than one signal mechanism is in effect to enhance the immune response against mannan [24]. The nature of protein–carbohydrate interactions has been largely satisfied using glycopolymer conjugates, which are evaluated in vitro for protein-specific interactions. Ooya et al. have studied the role of the molecular mobility in ligand multivalent interactions with specific proteins [33]. For this purpose ␣-cyclodextrin (CD)-encapsulated polyrotaxane based-polymers were synthesized in an effort to produce mobile CD-based ligands. Polyrotaxanes are macromolecules in which cyclic molecules such as CD can be threaded onto a PEO-like linear chain. PEO was used as a thread to produce supramolecular mobile CD-based structures, and Con A was selected as a binding protein for the study. Con-A-induced hemagglutination assay was used to determine the effect of multivalent interactions between Con A and maltoserotaxane conjugates. The data indicated that although the mobility of ligands is not the only factor, it has a direct impact on protein binding. Thus, it has been assumed that high mobility of maltose ligand possibly decreases the effect of nonspecific

APPLICATIONS

185

interactions between the ligand and Con A, thus preventing the entropic loss due to nonspecific binding and providing the entropy for specific interactions [33]. Some of the bioconjugation techniques of glycopolymers are mainly based upon the synthesis of relatively simple biological mimetics of complex saccharides, which are then found to either serve their biological functions upon their bioconjugation with other receptors or protein in vivo or are found to enhance the affinity of the ligands toward their receptors. Sulfation of glycopolymers is an important aspect of bioconjugation strategies and is found to play a dominant role in various biological phenomena. For example, glucosamineglycans (GAGs) are a type of sulfated saccharides found to play a dominant role in various physiological and pathological events. The synthesis of GAG-like polymers is a challenge due to the complexity of carbohydrate structures found in GAGs. Chondriton sulfate (CS) is a type of GAG that plays an important role in cell division, inflammation, and spinal cord injury. Rawat et al. have successfully synthesized CS-like carbohydrate structures using sulfated disaccharide monomers by ROMP. The biological activity of these polymers was then detected by measuring their ability to modulate neuronal growth, and it was found that neurite growth is significantly inhibited upon the treatment with these sulfated glycopolymers, and the inhibitory potency is comparable to those of natural GAGs. The discovery is consistent with previous studies that show that CS-coated surfaces can trigger downstream signaling that provides guidance for neuronal growth [34]. In addition to pathogen interactions on cell surface, protein–carbohydrate interactions are also found to play a dominant role in various physiological malfunctions that are not related to the pathogens [35]. Alzheimer disease (AD) is characterized by extracellular deposition of amyloid plaques in vulnerable brain regions. These plaques are made of small peptides (A␤) of 39–43 amino acids, and it is found that saccharides present on cell surface play an important role in the deposition of these peptides. One of the saccharides that play a key role in plaque stabilization is glucosamineglycans (GAGs). The further analysis of AD requires the study of interactions between A␤ and GAGs. The study of weak interactions between proteins and saccharides require synthetic glycoclusters to produce a glycocluster effect. The synthetic glycopolymers carrying bioactive saccharides have the potential to analyze AD in detail. Miura et al. have focused on the synthesis of sulfated glycopolymers to mimic GAG-like polymers to explore the mechanism of AD. For this purpose, sulfated glycopolymers of N-acetyl 6-sulfo ␣-d-glucosamine were synthesized by free radical polymerization and were incubated with A␤ peptide to study their interaction. It was found that glycopolymers with sulfated saccharides successfully inhibited amyloid formation after incubation with A␤ peptide, in contrast to nonsulfated saccharides where no change in amyloid formation was observed upon treatment with A␤ peptide [35]. In addition to GAGs, heparin-sulfate-based polymers are found to play an important role in regenerative responses upon their interaction with growth factors. These comparatively simple heparin sulfate mimics were prepared by cyanoxyl-mediated polymerization by Guan et al. These structures are found to act as an affective molecular chaperone for pro-angiogenic protein growth factor (FGF-2). These glycopolymers are not only found to impart resistance to the growth factor in response to degradation upon heat or enzymes but also promote the interactions of FGF-2 with

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GLYCOPOLYMER BIOCONJUGATES

its receptors. This capacity of synthetic sulfated glycopolymers is found to induce FGF-2-specific cell proliferation, suggesting their applications in angiogenesis and tissue engineering [36]. Another important application of polymer–protein bioconjugates is for drug delivery. These biohybrids—considered as new “chemical entities” with “regulatory agencies” as the biomolecules of interest—are covalently linked to the polymer at the surface rather than being encapsulated inside the polymer architecture. The attachment of drugs to protein–polymer conjugates in addition to increasing the specificity of the drug increases its solubility and molecular weight, thus reducing the renal clearance and increasing the circulation time in blood, which further increases the efficacy of a drug. ␤-Cyclodextrin-conjugated cancer drugs have been successfully prepared and are now in phase I clinical trials. The need is to further explore the field of glycopolymer bioconjugates, as these novel materials are found to possess various desired characteristics that can make them useful against many diseases and pathological disorders [37].

4.4 CONCLUSION AND FUTURE DIRECTIONS In short, glycopolymer bioconjugation strategies are explored in detail. The bioconjugation of carbohydrates to macromolecules was thought to be limiting and exhaustive due to the presence of limited functional groups on the surface of carbohydrates. With the advances in living radical polymerization, a variety of glycopolymer bioconjugates has been successfully prepared and has been utilized for various biological applications. Over a short period of time, the glycopolymer bioconjugates are found to dominate the fields of biomedicines, diagnostics, and biotechnology. The relative ease of synthesis of glycopolymer bioconjugates has also enabled the study of complex biological phenomenon including carbohydrate protein interactions. These polymer bioconjugates are used to understand the multivalent carbohydrate–protein interaction during biological events, thus providing a better understanding of biological phenomenon at the molecular level.

REFERENCES 1. Hermanson, T. G. (1996). Bioconjugate Techniques. Academic Press, pp. 1–15. 2. Lutz, F.-J., and Borner, G. H. (2008). Modern Trends in Polymer Bioconjugate Design. Prog. Polym. Sci. 33, 1–39. 3. Zhengwei, D., Lingshu, W., and Zhikang, X. (2008). Surface Glycosylation of Polymeric Membranes. Sci. China Ser. B–Chem. 51, 901–910. 4. Matsubara, T., Sumi, M., Kubota, H., Taki, T., Okahata, Y., and Sato, T. (2009). Inhibition of Influenza Virus Infections by Sialylgalactose-Binding Peptides Selected from a Phage Library. J. Med. Chem. 52, 4247–4256. 5. Gaek, M.-G., Stevens, C. R., and Charych, H. D. (2000). Design and Synthesis of Novel Glycopolythiophene Assemblies for Colorimetric Detection of Influenza Virus and E. coli. Bioconjug. Chem. 11, 777–788.

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24. De Graff, J. A., Kooijman, M., Hennink, E. W., and Mastrobattista, E. (2009). Nonnatural Amino Acids for Site-Specific Protein Conjugation. Bioconjug. Chem. 20, 1281–1295. 25. Deiters, A., Cropp, A. T., Summerer, D., Mukherji, M., and Schultz, P. G. (2004). SiteSpecific PEGylation of Proteins Containing Unnatural Amino Acids. Bioorg. Med. Chem. Lett. 14, 5743–5745 26. Geng, J., Mantovani, G., Tao, L., Nicolas, J., Chen, G., Wallis, R., Mitchell, A. D., Johnson, G. R. B., Evans, D. S., and Haddleton, M. D. (2007). Site-Directed Conjugation of “Clicked” Glycopolymers to Form Glycoprotein Mimics: Binding to Mammalian Lectin and Induction of Immunological Function. J. Am. Chem. Soc. 129, 15156–15163. 27. Rabuka, D., Forstner, B. M., Groves, T. J., and Bertozzi, R. C. (2008). Noncovalent Cell Surface Engineering: Incorporation of Bioactive Synthetic Glycopolymers into Cellular Membrane. J. Am. Chem. Soc. 130, 5947–5953. 28. Akasaka, T., Matsuura, K., and Kobayashi, K. (2001). Transformation from Block-Type to Graft-Type Oligonucleotide-Glycopolymer Conjugates by Self-Organization with HalfSliding Complementary Oligonucleotides and Their Lectin Recognition. Bioconjug. Chem. 12, 776–785. 29. Sun, X.-L., Faucher, M. K., Houston, M., Grande, D., and Chaikof, L. E. (2002). Design and Synthesis of Biotin Chain-Terminated Glycopolymers for Surface Glycoengineering. J. Am. Chem. Soc. 124, 7258–7259. 30. Umemura, M., Itoh, M., Makimura, Y., Yamazaki, K., Umekawa, M., Masui, A., Matahira, Y., Shibata, M., Ashida, H., and Yamamoto, K. (2008). Design of a Sialylglycopolymer with a Chitosan Backbone Having Efficient Inhibitory Activity against Influenza Virus Infection. J. Med. Chem. 51, 4496–4503. 31. Nagatsuka, T., Uzawa, H., Ohsawa, I., Seto, Y., and Nishida, Y. (2010). Use of Lactose against the Deadly Biological Toxin Ricin. ACS Appl. Mat. Interfaces 2, 1081–1085. 32. Rele, M. S., Cui, W., Wang, L., Hou, S., Barr-Zarse, G., Tatton, D., Ganou, Y., Esko, D. J., and Chaikof, L. E. (2005). Dendrimer-like PEO Glycopolymers Exhibit Anti-Inflammatory Properties. J. Am. Chem. Soc. 127, 10132–10133. 33. Ooya, T., Eguchi, M., and Yui, N. (2003). Supramolecular Design for Multivalent Interaction: Maltose Mobility along Polyrotaxane Enhanced Binding with Concanavalin A. J. Am. Chem. Soc. 125, 13016–13017. 34. Rawat, M., Gama, I. C., Matson, B. J., and Hsieh-Wilson, C. L. (2008). Neuroactive Chondroitin Sulfate Glycomimetics. J. Am. Chem. Soc. 130, 2959–2961. 35. Miura, Y., Yasuda, K., Yamamoto, K., Koike, M., Nishida, Y., and Kobayashi, K. (2007). Inhibition of Alzheimer Amyloid Aggregation with Sulfated Glycopolymers. Biomacromolecules 8, 2129–2134. 36. Guan, R., Sun, X.-L., Hou, S., Wu, P., and Chaikof, L. E. (2004). A Glycopolymer Chaperone for Fibroblast Growth Factor-2. Bioconjug. Chem. 15, 145–151. 37. Vicnet, M., Dieudonne, L., Carbajo, J. R., and Pineda-Lucena, A. (2008). Polymer Conjugates as Therapeutics: Future Trends Challenges and Opportunities. Expert Opin. Drug Deliv. 5, 593–614.

CHAPTER 5

GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES MARYA AHMED and RAVIN NARAIN Department of Chemical and Materials Engineering and Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, Canada

5.1 Introduction 5.1.1 Classification of Carbon Nanotubes 5.1.2 Structure of Nanotubes 5.2 Chemistry of Carbon Nanotubes 5.2.1 Shortening and Oxidation of Nanotubes 5.3 Functionalization of Nanotubes for Biomedical Applications 5.3.1 Noncovalent Approach 5.3.2 Covalent Approach 5.3.3 Hybrid Approach 5.4 Toxicity of Nanotubes 5.5 Applications of Carbon Nanotubes 5.5.1 CNT-Based Bioelectronics 5.5.2 Gene and Drug Delivery 5.5.3 Tissue Engineering 5.6 Conclusion and Future Directions References

189 190 190 192 192 193 193 196 204 206 206 206 207 207 209 209

5.1 INTRODUCTION Nanotechnology is defined as materials with at least one physical dimension in the range of 1–100 nm used to construct systems with novel properties [1–3]. The rapid progress in nanotechnology and nanoscience has led to the popularization of various Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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types of nanoparticles. Gold nanoparticles, magnetic nanoparticles, and quantum dots are just a few examples of spherical, metallic, and semiconductor nanoparticles that have been successfully synthesized and utilized for various biological applications including transport, imaging, and targeting [1, 3–6]. Furthermore, the unidimensional growth of materials to form nanowires and nanotubes with varying electronic and thermal properties has gained a lot of attention in recent years [2, 4, 7]. Carbon nanotubes (CNTs) are interesting examples of nanomaterials where targeting and tissue imaging capabilities are combined to produce multipurpose materials for biological, biomedical and pharmaceutical applications [1]. Carbon nanotubes are made up of carbon [3, 7], as long cylindrical fullerenes, which are composed of hexagonally arranged graphite structures and are capped at each end. These cagelike structures of carbon are found to possess exceptional material properties because of their unique structure. Some of these properties include their superior thermal and electronic properties, high elasticity, and tensile strength [2, 4, 6, 8–11]. The superiority of carbon nanotubes compared to other nanomaterials can be established by the fact that on one hand their thermal conductivity is twice as high as diamond and the electrical current-carrying capacity is 1000 times higher than that of copper wires, while on the other hand their tensile strength is reported to be 10–100 times higher than that of steel in the nanosized dimensions of the material [9]. 5.1.1 Classification of Carbon Nanotubes The discovery of carbon nanotubes has opened new venues for carbon nanotechnology [1]. However, CNTs cannot be classified as well-defined and organized molecules because of the inherent variability in their general structure. For example, CNTs vary in length and diameter and possess a range of helicities. The nanotubes prefer to aggregate in bundles of different diameters, and the probability of finding two identical nanotubes in the bundle is very small [3]. Thus, fractionation and purification of nanotubes is essential for their further characterization and utilization for biological applications [3]. The length, diameter, and chirality of carbon nanotubes further define their physical properties, including structural, electronic, and thermal properties [1]. Based on their general structure, carbon nanotubes are divided into two types: single-walled carbon nanotubes (SWNTs) made of single layer of carbon sheet and multiwalled carbon nanotubes (MWNT), where concentric cylinders of carbon come together to yield a tubelike structure [1]. These nanotubes are well-ordered graphitic material that are micrometers in length and 0.2–4 nm in diameter for SWNT and 2–100 nm in diameter for MWNTs. A brief overview of the structural differences of SWNTs and MWNTs is shown in Figure 5.1. These carbon allotropes are unidimensional and are found to have a high aspect ratio and high mechanical strength combined with a very light weight, excellent electronic properties, as well as chemical and thermal stability [8]. 5.1.2 Structure of Nanotubes Because of the facile synthesis of carbon nanotubes, this novel material has been largely used in research for its surface functionalization and utilization for various

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cm 1 Raman Spectrum of 90% SWNT

Structure

Electronic microscopy

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0

0

Wavenumber (cm 1)

FIGURE 5.1 Schematics explaining the difference in structure of SWNTs and MWNTs using microscopic and spectroscopic techniques. (Reproduced with permission from Ref. [40].)

biomedical purposes [4, 6, 8]. The general preparation of CNTs involves three methods: laser ablation, arc discharge, and chemical vapor deposition. All these methods produce CNTs with residual catalysts or other impurities, and further purification of nanotubes is required before their utilization for biomedical uses [1]. Now a brief overview of the general structure of single-walled carbon nanotubes will be provided. The theoretical structure of SWNTs can be defined with the help of the roll up vector r and the chiral angle . The roll-up vector r can be defined as a linear combination of base vectors a and b of a hexagon [4]: r = na + mb where m and n are integers and r is perpendicular to the axis of the nanotubes. The values of m and n differ by the change in diameter of the nanotubes, and hence nanotubes with varying sizes will have different electronic configuration and conductivity [4]. SWNTs were first reported in 1993. Since then exceptional work has been done on their surface functionalization and characterization. These nanotubes are found to have potential for a variety of applications. However, SWNTs in their native state contain various impurities, including amorphous carbon and metal residues. The electronic transitions between the energy bands of nanomaterials can be studied by using various spectroscopic techniques. The ultra-high-vacuum scanning tunneling microscopy has revealed that SWNTs can behave as metals and semiconductors depending upon their diameter and chirality as determined by the study of the

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measurement of electronic band structure. The diameter of nanotubes is found to be inversely proportional to the band gaps, thus predicting the relationship between the size and conductivity of nanotubes, which coincides with theoretical prediction [10].

5.2 CHEMISTRY OF CARBON NANOTUBES The reactivity of nanotubes is mainly determined by their spherical geometry, and carbon nanotubes are expected to be more reactive than a plain sheet of graphene due to the curvature-induced strain of carbon atoms and misalignment of the ␲ orbitals in the tubelike morphology compared to a plain sheet itself, which can partially be relieved by reacting with other molecules present on the surface. The study of the chemistry of CNTs can divide nanotubes into two distinct regions: open ends and side walls of nanotubes. Open ends of nanotubes can be produced by fragmenting the closed-end or pristine nanotubes using different techniques such as acid treatment and ball milling and are more prone to surface functionalization than closed-end nanotubes. The functionalization of open-end nanotubes with exterior walls (convex surface) and interior wall (concave surface) is important in tailoring the properties of these materials [12]. The chemical modification of nanotubes along with maintaining the basic electronic structure is a major issue in the surface functionalization of these materials. Another important aspect about the chemistry, functionality, and solubility of nanotubes is the purity of the material. This is critical for the use of nanotubes for further applications. However, this aspect has received limited research attention. The nanotubes as received are contaminated by various impurities, and their surface functionalization largely depends upon the type of impurities present in the sample. All biological, biochemical, and chemical processes occur in solution. Hence dissolution of nanotubes in a suitable solvent is the most important step toward their surface functionalization. Nanotubes normally exist as ropes or bundles 20–25 nm in diameter and a few microns in length and are extremely resistant to wetting. In addition these bundles tend to entangled with each other, producing a highly dense complex network structure that is not dispersible in organic solvents [10]. 5.2.1 Shortening and Oxidation of Nanotubes One way to disperse nanotubes in solution is treatment with strong acids that in addition to purify them produce oxidizing groups on the open ends. These acidpurified nanotubes can now be dispersed in a variety of solvents. However, this treatment does produce some defects in the structure of the CNTs. These defects include disruption of electronic structure, producing holes in the structure of CNTs and producing impurity states. The holes produced in CNTs can then be used to shorten CNTs during the oxidization process. The shortened nanotubes are more dispersible in solvents compared to the longer ones. Oxidative doping is one of the various methods to functionalize the surface of nanotubes with oxidative groups. The aromatic ring system of nanotubes gets disrupted upon their exposure to strong chemical agents. The sonication of single-walled nanotubes in strong acids such as HNO3 and H2 SO4 helps functionalize the nanotubes with COOH groups and quinines,

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while sonication of these nanotubes in the presence of organic solvents produces dangling bonds that can then be used for further modification purposes. The extent of surface modification of nanotubes is dependent upon their diameter as determined by the band-gap energies of nanotubes, thus larger diameter nanotubes require more harsh conditions for surface functionalization compared to smaller ones [10]. The full-length nanotubes in contrast are ideal materials to form composite nanoscale structures. The dissolution process of nanotubes can also be accomplished by direct reaction of COOH-functionalized nanotubes with amine-containing polymers, and this ionic stabilization is found to be advantageous for the following reasons: (1) the acid–base reaction seems to be ideal to disperse nanotubes in the solution without destroying the length of nanotubes; (2) compared to covalent bonding, ionic bonds present on the surface of nanotubes can be easily exchanged with the molecule of choice; and (3) such ionic interactions further allow interactions with biomolecules such as DNA (deoxyribonucleic acid), thus allowing the synthesis of biocompatible nanotubes. Although oxidation of nanotubes is the most widely used methods for surface functionalization approaches, other functionalization techniques like doping of nanotubes with halogens are also used in the absence of acidic environment, thus preventing the oxidative damage and shortening of nanotubes in acidic conditions. The functionalization of nanotubes by Br, F, or I can further facilitate their functionalization with molecules of choice [10, 12].

5.3 FUNCTIONALIZATION OF NANOTUBES FOR BIOMEDICAL APPLICATIONS The major limitation of nanotubes for biological and biomedical applications is their solubility in an aqueous media [8]. The solubility issues of these nanotubes are resolved by using various polymers and biomaterials, and their surface modification is detected using absorption spectroscopy and photoluminescence to investigate the interband optical transition of chiral CNTs [10]. Next to genome and proteome, carbohydrates are the third information-carrying molecule in nature that play an essential role in cell growth, differentiation, cell–cell communication, and immune recognition [13]. Recent demonstrations have shown that biomolecules such as peptides, protein, nucleic acids, and carbohydrates can be used to surface functionalize the nanotubes. This has opened up new venues for carbon nanotechnology in biomedicines. In addition to providing solubility in aqueous media, these natural macromolecules are also found to increase the biocompatibility of carbon nanotubes in living tissues. The focus of this chapter will be the synthesis and characterization of glycopolymer-functionalized nanotubes using various strategies [8]. The immobilization of biomolecules on the surface of nanotubes has been achieved by three methods: (1) noncovalent approach, (2) covalent approach, and (3) hybrid approach [8]. 5.3.1 Noncovalent Approach Noncovalent functionalization deals with surface wrapping of nanotubes with macromolecules as well as with neutral hydrophilic polymer chains. This facile approach

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for nanotube functionalization deals with the incubation of natural glycopolymers with a nanotube solution. For example, the coating of chitosan on SWNTs was obtained using ultrasonic dispersion for 60 min in an ice bath. The obtained solution was ultra-centrifuged to remove the bundles of SWNTs, and the supernatant was analyzed for further analysis of SWNTs. However, this method largely limits the control over the tailoring and surface functionalization of the nanomaterials synthesized [14]. Amylopectin is another example of a naturally occurring branched glycopolymer that has been used for the surface functionalization of nanotubes due to its unique conformation in solution. Amylopectin consists of 8–12 outer branches of glucose residues and can attain coiled conformation upon exposure to iodine solution or in the presence of hydrophobic structures such as nanotubes. The wetting of SWNTs in an amylose solution is another way to functionalize the surface of these nanomaterials and to improve their solubility in aqueous solution. Briefly, the solution of nanotubes was sonicated in amylose solution and the resulting solution was decanted and dried to yield functionalized nanotubes [15]. Schizophyllan (SPG) is a naturally occurring glycan that has been largely used for many biological application including gene and drug delivery and for surface modification of nanomaterials. To obtain a supramolecular structure of SWNTs wrapped in carbohydrates, SPG–lactose complexes were dissolved in dimethyl sulfoxide (DMSO). The solution was mixed with an SWNT aqueous solution, and a supramolecular assembly of SPG–lactose on the SWNT surface was confirmed using infrared (IR) and ultraviolet (UV) visible (VIS) spectroscopy. The mechanism of the assembly of this biomolecule-stabilized nanostructure is as follows. SPG exists in a triple helical and random coiled single-stranded state depending upon the type of solvents used for its dispersion. For example, in DMSO, SPG exists in a singlestranded states. However, when this solution of SPG is dispersed in water containing SWNTs, a phase change of biomolecules from single-strand to triple-helix state occurs, wrapping around the surface of nanotubes to produce biocompatible structures. Lactose-functionalized SPG was used where SPG itself served as a wrapping agent to shield the hydrophobic nature of nanotubes, while lactose moieties functioned as appendages that can independently perform their biological functions, thus making the macromolecule suitable for various biomedical applications. The lactosefunctionalized SPG-SWNTs were further utilized for lactose affinity purposes and the process was studied using atomic force microscopy (AFM) [16]. Another strategy to surface functionalize nanotubes via a noncovalent approach to maintain their novel inherent properties along with increasing their solubility and biocompatibility is coating of nanotubes with biomolecules via wet spinning. Lynam et al. have used a variety of natural polysaccharides including chitosan, hyaluronic acid, heparin, and chondriton sulfate to shield the surface of nanotubes, and all these biomolecule-functionalized scaffolds are found to promote cell growth providing a potential platform for tissue engineering applications [17]. The combination of conductivity of CNTs along with physiological properties of biomolecules provides an ideal approach for the growth of living cells. The coating of biomolecules on the surface of CNTs was simply obtained by sonicating the nanotubes in a solution

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of biomolecules of appropriate concentrations. The concentration of biomolecules higher than optimum value resulted in aggregation of nanomaterials in solution. In an effort to obtain polymer-coated nanotubes via the wet spinning method, a coagulation bath containing a biocompatible polymer such as heparin along with charged molecules such as DNA or chitosan was used, and the solution of SWNT was dispersed in a coagulation bath solution with the help of a syringe. The mixture was spinned at a constant rate to obtain polymer-spinned nanomaterials. Experimental factors such as speed of spinning and injection rate were found to be crucial for the desired results. Electrochemical characterization of these nanofibers revealed that nanotubes maintain their potential as conductive materials after coating with insulating biocompatible layers [17]. Amylose-like amylopectin is another interesting polysaccharide for the surface functionalization of nanotubes because of its ideal helical conformation upon exposure to long hydrophobic segments. Amylose in this conformation contains 6–8 glucose units per turn with a cavity diameter from 0.54 to 0.97 nm, thus producing a hydrophobic channel with hydroxyl groups pointed toward the outside. The hydrophobic interaction between the helical channel of amylase and CNT surface is the driving force for the stabilization of nanotubes. The amylose molecules wrap the nanotubes through favorable hydrophobic interactions during the noncovalent immobilization technique. The wrapping of amylose on the surface of nanotubes was first obtained through the helical conformation of amylose in the presence of iodine. The subsequent dispersion of nanotubes in this solution displace iodine molecules inside the helix to produce a biomolecule-stabilized nanostructure. However, the major issue in this regard was steric hinderance during complex formation between SWNTs and helical conformation of amylose structure [18, 19]. To solve this problem and to further improve the dispersion stability of nanotubes in aqueous solution amylose-f -SWNTs were prepared through vine-twining polymerization. The enzymatic polymerization of maltohepatose was done in the presence of SWNTs and enzyme phosphorylase. It was found that pristine SWNTs were insoluble and exist as entangled and agglomerated bundles in aqueous solution. In contrast, after enzymatic polymerization the complex structure of nanotubes became separated into individual bundles. Raman spectra of pure and functionalized nanotubes was obtained and was compared to prove the surface functionalization of nanomaterials [20]. It should be noted that this surface functionalization technique is a type of noncovalent approach where in situ polymerization was performed to stabilize the glycopolymer on the surface of nanotubes. These noncovalent approaches for surface modification of nanotubes are indeed advantageous as they do not alter the surface properties of nanotubes during the functionalization process. However, the process greatly limits the choice of macromolecules and polymers that can be utilized for this purpose. Moreover, the higher concentrations of these macromolecules are required for coating purposes, thus limiting the functional properties of coated nanotubes. The nanocomposites formed are not found to be stable over longer periods of time and also inhibit further functionalization of the material [21]. One way to solve these problems associated with the noncovalent approach is covalent functionalization, which in turn has its own advantages and drawbacks [21].

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5.3.2 Covalent Approach The covalent surface functionalization of carbon nanotubes with biomolecules of choice requires the knowledge of the basic bioconjugation strategies. The most common method for covalent functionalization of nanotubes involved COOH residues on the surface of CNTs, which are introduced by the oxidation of pristine nanotubes with strong acids. The location of COOH after the treatment exists predominantly at the open ends and the defective regions of nanotubes, compared to the side walls. For side-wall functionalization other reactions including nitrene cycloaddition, arylation using diazonium salts, and 1,3-dipolar cycloaddition are employed, and various proteins, peptides, and nucleic acid molecules are immobilized on the surface of nanotubes using these techniques. Although excessive oxidation can alter some of the properties of nanotubes and the immobilization approach is nonspecific, the coupling of biomolecules using COOH group chemistry is one of the simplest and most widely used bioconjugation strategies for nanotube functionalization. In contrast, side-wall modification methods are more specific and less disruptive to the inherent properties of nanotubes. However, they do require complex organic chemistry techniques to immobilize the materials on the surface of nanotubes [8]. The integration of CNTs into the polymer matrix is found to have numerous applications, including the development of nanomaterials with extraordinary mechanical properties [2, 11]. The covalent functionalization of polymer on the surface of nanotubes can be done using two approaches: grafting-to or grafting-from approach. Grafting to involves the direct conjugation of polymers on the surface of nanotubes with the help of functional groups present on both polymers and the convex surface of CNTs. Hence, the approach is limited by the presence of reactive functional groups on both moieties and high steric hindrance of macromolecules. In contrast grafting from involves growth of polymers from the surface of CNTs by in situ polymerization of monomers on the initiating sites anchored on the surface of CNTs. The living radical polymerization (LRP) techniques have further improved the use of the grafting-from approach for this purpose. Grafting the polymers on the surface of nanotubes can be done using various strategies, including click chemistry, reversible addition fragmentation chain transfer (RAFT), anionic polymerization, ␥ -ray irradiation, polycondensation, electrochemical grafting and atom transfer radical polymerization (ATRP). ATRP is a very efficient and facile technique, and a variety of ATRP-active monomers including methacrylates, acrylamides, and styrenes have been grafted on to macroinitiators-modified nanotube surfaces using ATRP. It has been found that grafting density on the surface of CNTs can further be controlled by changing the feed ratio of monomer to macroinitiator-functionalized CNTs. Grafting water-soluble polymers on the surface of CNTs are of great interest due to their ability to increase the solubility of CNTs in water. The grafting of biocompatible, water-soluble glycopolymers on the surface of MWNTs via in situ polymerization has been reported by ATRP of 3-O-methacryloyl1,2:5,6-di-O-isopropylidene-d-glucofuranose (MAIG), as shown in Scheme 5.1. The process uses CNT-based macroinitiators to initiate the polymerization process on the surface of nanotubes. The glycopolymer-functionalized CNTs thus produced

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O O O

O

H

[

MWNT

O

OH

O ]k

Sn(Oct)2

O

O

n O

120°C

SCHEME 5.1 Grafting of PCL on the surface of MWNTs. (Reproduced with permission from Ref. [26].)

were further deprotected using 80% formic acid to yield polyhydroxy MWNT-gpolyMAIG. This step of deprotection was confirmed by proton nuclear magnetic resonance (1 H-NMR) and fourier transform infrared (FTIR). Furthermore, it was noted that polyMAIG-g-MWNTs were soluble in apolar and weakly polar solvents such as chloroform, tetrahydrofuran (THF) and ethylacetate. In contrast deprotected CNTs were not soluble in nonpolar solvents and were highly soluble in polar solvents such as water. The morphology of these nanotubes was obtained by scanning electron microscopy (SEH) and transmission electron microscopy (TEM) images that indicated the presence of continuous polymer phase on the surface of nanotubes compared to nonfunctionalized ones. Scanning force microscopy (SFM) is another powerful tool to detect individual brushes on an immobilized surface. SFM images of polymer-modified MWNTs compared to nonfunctionalized ones showed that, although the cylindrical morphology was maintained for Br-functionalized MWNTs in both height and phase images, polymer-functionalized images revealed different morphology where nanotubes were found to possess fuzzy and collapsed structures, indicating the higher density of polymer brushes on the surface [22]. Narain et al. have also employed this grafting-from approach to surfacefunctionalized CNTs with two different biocompatible polymers [23]. For this purpose carboxyl-group-functionalized nanotubes were activated with thionyl chloride to produce hydroxyl-functionalized nanotubes that were then modified with an ATRP initiator, bromo-isobutyryl bromide, for their surface functionalization with polymers by ATRP (Scheme 5.2). The polymerization of glycomonomer lactobionamidoethyl methacrylate (LAMA) and methacryloyloxyethyl phosphorylcholine (MPC) on the surface of SWNTs was carried out by ATRP as discussed above. The reaction was carried out in the absence of protected group chemistry, thus bypassing several steps of protection and deprotection of hydroxyl groups on the surface of carbohydrates. These glycopolymer- and MPC-functionalized SWNTs have potential for various biomedical applications [23]. The cationic glycopolymer-functionalized nanotubes were later used for gene delivery purposes and were found to have enhanced gene transfer ability with comparatively low toxicity [24]. Generally, long polymer chains are grafted on the surface of nanotubes by the grafting-to approach using amidation and esterification reactions. The major

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O

O

SOCI2 65°C

OH

O

OH

H2N

N H

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Br

TEA, THF

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O

Monomer N H

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CuBr, bpy, 25°C

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O Br

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O

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O M:

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HO

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OH TEA

N H

O

Br N(CH2CH3)3

O Excess TEA O N H

+

O

+

–

HN(CH2CH3)3-Br

O

SCHEME 5.2 Covalent functionalization of biocompatible polymers on the surface of nanotubes by ATRP in the absence of protected group chemistry. (Reproduced with permission from Ref. [23].)

challenge in this regard is to modify the surface of nanotubes with reactive functional groups. Maleic anhydride is a highly reactive and versatile functional group that can be used to conjugate a variety of polymers and peptides. Hong et al. have surface-functionalized nanotubes with long-chain poly(styrene-alt-maleic anhydride) P(St-alt-MAh) in an effort to preserve the unique properties of nanotubes, in conjunction with grafting a higher density of functional groups on the surface of nanotubes [21]. P(St-alt-MAh) is a synthetic polymer that contains a large number of maleic

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anhydride functional groups in the main chain. In addition this polymer has a potential for further conjugation with a variety of macromolecules. The in situ functionalization of MWNTs with P(St-alt-MAh) was performed in multisteps. The crude nanotubes obtained were oxidized with HNO3 using an ultrasonication process to generate MWNT-COOH, which were then used to immobilize chain transfer agent sites on the surface of nanotubes for their use during RAFT polymerization. The MWNTCOOH was reacted with thionyl chloride to obtain MWNT-COCl, which was reacted with 2-hydroxyethyl-2-bromoisobutyrate to yield MWNT-Br. The MWNT-Br was further modified to yield dithioester-functionalized MWNTs using phenyl magnesium bromide and carbon disulfide, as shown in Scheme 5.3 and MWNT-SC(S)Ph was obtained after purification. The dithioester-functionalized MWNTs served as a chain transfer agent during RAFT polymerization to grow the polymeric chains on the surface of nanotubes during in situ polymerization [21, 25]. Compared to other methods of polymerization, the RAFT method is versatile and tolerant toward different types of monomers, solvents, and experimental conditions [25]. The in situ RAFT polymerization of St and MAh was performed in THF, yielding P(St-alt-MAh)-f -MWNTs, and these nanomaterials were further characterized using thermogravimetric analysis (TGA) and FTIR to confirm the presence of functional polymer chains on the surface of nanotubes. The presence of a number of reactive functional groups on the surface of nanotubes is crucial for its further modification. The 1 H-NMR spectrum was performed to determine the quantity of anhydride groups present on the surface of nanotubes. The unique structural and electronic properties of nanotubes make them a potential candidate for numerous biological applications, including biosensors, scaffolding for cell growth, transporters for gene and drug delivery, and imaging. However, their hydrophobicity and nonbiocompatibility are major limitations in this regard. From maleic anhydride sites, MWNTs can react with OH- and NH2 -functionalized polymers. Thus amino sugar (1-amino-1-deoxy-␤-dlactose) was conjugated on the surface of nanotubes by the grafting-to approach to further improve its biocompatibility for living systems, and the conjugation process was further confirmed using FTIR (Scheme 5.4) [21]. Poly(ε-carpolactone) (PCL)-functionalized MWNTs were also prepared by the grafting-to approach and their biodegradability was studied. The first step toward

O O

O O O O

(

(

O

O

Br O

hydrolysis

O O

n O

O O

MWNT-g-polyMAIG

HCOOH

k

CuBr/HMTETA, 60°C

k

k O

MWNT-Br

O

Br

Br

(

O

O

(

(

(MAIG)

(

O

O

O n O

O

O

O

O O O

MWNT-g-polyMAG

HO

OH OH O

HO

SCHEME 5.3 Grafting linear glycopolymers from surfaces of MWNTs by ATRP. (Reproduced with permission from Ref. [22].)

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GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

CH3 COOH

(a) SOCl2

COOCH2CH2OCOC

HNO3

Br

CH3 CH3 (b) HOCH2CH2OCOC Br CH3

1

2

3 CH3

S

COOCH3CH3OCOC S C CH3

S C

S MgBr

4

SCHEME 5.4 Stepwise functionalization of nanotubes with RAFT agent: (1) Pristine nanotubes, (2) Carboxyl-functionalized nanotubes, (3) halide-functionalized nanotubes, and (4) RAFT agent-stabilized nanotubes. (Reproduced with permission from Ref. [21].)

this process was the introduction of initiating sites on the surface of MWNTs. For this purpose, oxidized MWNTs (MWNT-COOH) were first reacted with thionyl chloride and then with glycerol to yield hydroxyl-functionalized MWNTs (MWNTOH). The density of hydroxyl groups on the surface of MWNTs was determined using TGA, and PCL chains were then grafted on the surface of MWNTs by in situ ring opening polymerization of ε-carpolactone in the presence of catalyst as shown in Scheme 5.5 [26]. Attaching carbohydrate to the surface of MWNTs allows the development of novel glyconanotechnology, which can be utilized for tissue targeting purposes and for gene and drug delivery. The galactose-functionalized MWNTs were prepared by Jain et al. to enhance their solubility and for their use in biological applications. In this context amine-functionalized nanotubes were prepared by acylation. Acylated MWNTs produced by oxidation and thionyl chloride reaction were further reacted with ethylene diamine (EDA), and the number of free amine residues on nanotube surfaces were quantified using the Kaser test. The amine-functionalized MWNTs thus obtained were used to conjugate d-galactose. During this process of galactosylation, acidic pH promotes the ring opening of sugar molecules, which facilitate their conjugation with free amine of MWNTs. These galactose-conjugated MWNTs have the potential to serve as targeted drug delivery vehicles [27]. The covalent functionalization of nanotubes with biomolecules is crucial to control the specificity of the reaction in order to shape the applications of novel nanomaterial

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FUNCTIONALIZATION OF NANOTUBES FOR BIOMEDICAL APPLICATIONS

St + MAh RAFT Polymerize

CH3 S COOCH2CH2OCOC S C

O

CH3 OH

O CH3O

O

O

n

OH , HO

OH O HO

O HO

O OH

NH3

SCHEME 5.5 Conjugation of glycopolymers to RAFT-functionalized nanotubes. (Reproduced with permission from Ref. [21].)

synthesized. The electrochemical sensing platform was established upon the integration of redox mediators and CNTs in a chitosan matrix. These embedded CNTs were used to help the redox mediators in facilitating the flow of electrons in electrochemical devices such as biosensors, fuel cells, and reactors. For this purpose CNTs were solubilized in aqueous solution with the help of chitosan, followed by the casting on the surface of a glassy carbon electrode. The main criteria to produce these novel biosensors is the long-term stability of chitosan-stabilized nanotubes, which can be obtained using the covalent approach for surface functionalization. Chitosanmodified CNTs with improved storage modulus and water solubility are successfully prepared by a number of techniques, including covalent stabilization of chitosan on the surface of nanotubes. Shieh and Yang used a layer-by-layer deposition technique along with the covalent approach to synthesize nanocomposites of desired characteristics [28]. For this purpose chitosan was grafted on the surface of nanotubes by the amidation reaction between carboxyl groups of CNTs and amine groups of chitosan

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GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

at 98◦ C for 24 h in an acidic environment, and the extent of grafting was determined using TGA. The chitosan-grafted CNTs were dissolved in an acidic solution without further purification, and a chitosan solution of varying concentration was added to the mixture followed by casting into thin films. It was found that chitosan-grafted CNTs and chitosan solution when mixed in a ratio of 1 : 1 provided optimum results for the uniform distribution and alignment of nanotubes in the matrix. These films were dried under vacuum at 60◦ C. It was thought that chitosan-grafted CNTs will be miscible with the chitosan matrix and will further allow a higher degree of CNTs incorporation without phase separation. The process will further improve the mechanical properties of nanotubes as it is expected that chitosan grafted on the surface of nanotubes can become physically entangled with chitosan chains in solution, thus providing strong interactions between chitosan and nanotubes. In the design of this nanocomposite, CNTs will serve as a network to distribute the stress evenly on the polymer matrix and improving the overall mechanical properties of the composite [28]. The covalent functionalization of CNTs with macromolecules of choice possesses various advantages compared to noncovalent techniques. For example, a broad range of polymers can be used for the surface functionalization of nanotubes, linkage between nanotube–polymer complexs is more stable and effective, and nanocomposites obtained by covalent functionalization exhibit higher solubility even in the presence of a low degree of functionalization. The versatility of available bioconjugation techniques allows flexible and efficient synthetic routes to synthesize the nanocomposites of desired functionalities. Most of the synthetic polymers used for this purpose contain aromatic rings, which incorporate the toxicity to the resultant products, thus limiting their applications for biomedical applications. Chitosan, a copolymer of 2amino-2-deoxy-␤-d-glucopyranose and 2-acetamido-2-deoxy-␤-d-glucopyranose is cationic in nature and is the second most abundant natural polymer on earth. Due to their biocompatibility and biodegradability, chitosan polymers are found to have numerous applications in medicine, pharmaceutics, antimicrobial agents, and in various other fields. One of the interesting applications of chitosan is its metal-chelating ability. Because of this property, these polymers are largely used for effective removal of pollutants. Similarly, CNTs are also shown to possess efficient metal removal properties due to the structure and properties of CNTs. Thus combination of CNTs and chitosan is a promising approach to develop environmentaly friendly nanocomposites, containing potential applications in catalysis and environmental protection. Pristine MWNTs range from micrometers to millimeters in size, and they are highly tangled and insoluble in nature. The long and tangled nanotubes can be converted into shortened nanotubes by a cutting approach so that they can be suspended in the solvents and can be modified by the molecule of choice [29]. To functionalize MWNTs with low-molecular-weight chitosan, the MWNTs were first shortened using a ball-milling method and were then acid treated to generate the functional groups on the surface of nanotubes. MWNT-COOH was reacted in the presence of thionyl chloride to yield MWNT-COCl, which further reacted with amine of chitosan to yield chitosan-f -MWCNTs. The nanocomposites produced were extensively washed and dialyzed to remove the adsorbed chitosan, and the final product was analyzed using

FUNCTIONALIZATION OF NANOTUBES FOR BIOMEDICAL APPLICATIONS

203

FTIR. TGA showed the polymer component present on the surface of MWNTs was about 58% [30]. Cellulose acetate (CA) is one of the derivatives of cellulose that is found to have various applications in the fields of drug delivery, antimicrobial agent synthesis, and in the food industry. MWNTs were functionalized with cellulose acetate after ball milling, extensive purification, and oxidation using acids as discussed before. Amine-functionalized MWNTs were then prepared by refluxing with acylchloride and reacting the resultant products with triethyl amine, and 1,3-propanediamine in acetone, and the mixture was stirred under nitrogen for 48 h. The solvent was removed and nanotubes were purified by extensive washing. MWNT-NH2 was then reacted with 2,4,6-trichloro-1,3,5-triazine for 48 h and the black residue was obtained by filtration and was further purified to eliminate the excess of triazine. Further functionalization of MWNT-triazine with CA was carried out at 100◦ C for 48 h and the mixture was filtered using a nylon membrane. The excess of CA was removed and the final product was dried under vacuum [31]. The polymeric and monomeric molecules have extensively been utilized for the surface functionalization of nanotubes. In the field of bioscience various carbohydrate-stabilized nanomaterials have been prepared in an effort to mimic the extracellular structure of living cells and to study their molecular interactions. To achieve glycoconjugate-modified nanotubes, glycomonomer p-Nacryloylamidophenyl-␣-d-glucopyranoside was polymerized via a free radical polymerization technique and the polymer was purified by dialysis. The SWNT solution was added to the solution of a polymer, and the suspension was homogenized by sonication using a tip-type sonicator for 2 h. The black solution was centrifuged at 10,0000g for 1 h. The carbohydrate–CNT conjugates can be used for recognition purposes if the carbohydrates attached to the surface of CNTs are monomeric subunits and can provide a multivalent effect. Hence, compared to natural glycopolymer of complex structure, synthetic glycopolymer conjugates bearing scaffolds can serve as ideal candidates for this purpose. Furthermore, glycoconjugate polymers form a large helix of the main chain with hydrophilic brushes, which facilitates the dissolution of nanotubes in water. The dispersion is obtained by hydrophobic interactions between polymers and nanotubes with an outer core of sugar residues. These densely oriented sugar residues serve as multivalent carbohydrate ligands with potential biological applications [32]. The functionalization of nanotubes with glycomonomer-based initiators is another approach to produce glycopolymer composites. The first step was purification and oxidation of SWNTs in acidic conditions as discussed earlier. The SWNTs obtained were filtered, dried, and characterized using various techniques. The Raman spectra of raw CNTs show a distinct absorption peak around 1350 cm−1 , which is attributed to the disordered C–C bonds of amorphous carbon. The purification of SWNTs was indicated by the disappearance of this absorption peak after treatment with HNO3 . The further incubation in an acidic environment drastically changed the Raman spectra, and the appearance of another peak indicated the presence of carboxyl groups. This procedure was used to improve the wetability of nanotubes for a short period

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GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

of time. Hence further modification was required for the long-term solubility of nanotubes in an aqueous solution. To modify the SWNTs using pMEGlc macroinitiator, SWNT were dispersed in a pMEGlc solution and the mixture was incubated under nitrogen at 70◦ C for 24 h. The solution was evaporated, and SWNT was purified by repeated washing, and functionalization of nanotubes was further confirmed with Raman spectra. The SWNTs contain a large amount of ␲ electrons, which tend to trap radicals of varying sizes, thus resulting in macroinitiator-stabilized nanotubes. The biological activity of polymer-modified SWNTs was detected by mixing pMEGlcmodified SWNTs with concanavalin A (Con A), and precipitates were observed upon the incubation of nanotubes with a Con A solution. It was found that specific adsorption of Con A on glycopolymer-functionalized nanotubes occurs. Thus glycopolymers maintain their biological properties after their adsorption on the surface of nanotubes [33]. 5.3.3 Hybrid Approach The large aromatic groups present on the hydrophobic surface of CNTs make them ideal for the noncovalent interactions with macromolecules. This strong noncovalent interaction between macromolecules and CNTs is also employed by researchers for their further functionalization with biomolecules, and the process is termed hybrid approach. During this process, noncovalent adsorption of aromatic chains containing functional groups is first accomplished, and the functional groups are then covalently linked to biomolecules of choice. A major advantage of combining covalent and noncovalent strategies into a hybrid approach is that immobilization of macromolecules occurs without disrupting the native structure of nanotubes. However, these techniques are also accompanied with some drawbacks, including lack of specificity and denaturation of macromolecules upon adsorption [8]. A variety of approaches have been utilized to produce chitosan-modified CNTs, as discussed above. The noncovalent adsorption of chitosan on the surface of nanotubes suffered from long-term stability problems, and covalent approaches in contrast were accompanied with the risk of altering the electronic properties of nanotubes. To yield stable and noncovalently modified chitosan-functionalized CNTs, pyrenefunctionalized chitosan derivatives were prepared and were further utilized for noncovalent modification of CNTs. Pyrene is a fluorescent compound that in addition to providing the fluorescent properties to nanomaterials serves as an excellent moiety to interact with the ␲ bonds on the surface of CNTs, as shown in Scheme 5.6. This process of noncovalent modification was further confirmed by 1 H-NMR spectroscopy, which showed the shift and broadening of peaks associated with pyrene molecules due to their interactions with ␲-␲ stacking of CNTs. Although amine of chitosan is also known to strongly interact with CNTs, due to the electron-donating property of the former, the reaction was carried out in acetic acid solution, hence suppressing this election-donating property of chitosan. Furthermore, conjugation of pyrene at the amine moiety of chitosan enhances the possibility of interaction of pyrene with the surface of CNTs compared to residual secondary amine due to steric hindrance of bulky pyrene groups. The modification of CNTs with fluorescently labeled

FUNCTIONALIZATION OF NANOTUBES FOR BIOMEDICAL APPLICATIONS

OH

HO O

NHCOCH3

O O

HO

NH2

205

O

OH

n

O

O

O N Aqueous acetic acid + DMF

O

OH

HO O

NHCOCH3

O O

HO

NH

O

OH

O

n

π-π Stacking in aqueous acetic acid

OH

HO O

NHCOCH3

O O

HO

NH O

OH

O n

SCHEME 5.6 Functionalization of nanotubes with glycopolymers using hybrid approach. (Reproduced with permission from Ref. [11].)

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GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

chitosan was further confirmed using fluorescence spectroscopy. The results clearly showed that although pyrene-labeled chitosan was fluorescent, no fluorescence was detected for the pyrene-labeled chitosan-modified CNTs, possibly due to the disruption of ␲ conjugation due to conformational change in the pyrene structure. In addition, where pristine CNTs were completely insoluble in aqueous solution due to the strong hydrophobic structure of graphite, polymer-stabilized CNTs were found to be well soluble in aqueous solution. The effect of temperature on the stability of polymer-functionalized CNTs was further studied, and it was found that upon higher temperatures, precipitation of nanotubes in aqueous solution occurs due to the very low binding energy between pyrene and CNTs (∼55 pN) [11].

5.4 TOXICITY OF NANOTUBES The aqueous solution of nonfunctionalized CNTs is known to be unstable in water. It was also found that noncovalent modification of nanotubes largely improves their stability, the process is dependent upon the type of macromolecule used to stabilize the surface. With the development of covalent approaches to modify the surface of nanotubes and the stable dispersion of nanotubes in aqueous media, toxicity of these nanomaterials became an important issue. The toxicity studies are being done as a function of type (SWNTs versus MWNTs), length, surface functionalization, concentration, and degree of dispersion. However, very limited data exists, and there is considerable disagreement in the literature about the toxicity of these materials. Several hypothesis can explain the possible pathways of cytotoxicity of these materials. These include degree of purity of sample, physical contact between nanotubes and cells, and production of reactive oxygen species upon exposure to these nanomaterials [34]. In addition it was also reported that type of toxicity assay used for the detection might exert some influence on the toxicity data [35]. Hence more comprehensive studies are required to evaluate the effect of these novel materials on biological functions.

5.5 APPLICATIONS OF CARBON NANOTUBES The unique properties of CNTs, including high mechanical strength, high surface area, and smaller size, make them ideal for a variety of biomedical applications. Although detailed synthesis and utilization of glycopolymer-functionalized nanotubes have been discussed above, a brief overview of some examples of CNT-based applications are given below. 5.5.1 CNT-Based Bioelectronics Biomolecule-functionalized nanomaterials have the potential to serve as a new generation of biosensors and improved performances over other technologies. Various protein-based CNTs were successfully prepared and were utilized as potential biosensors. A glucose biosensor was prepared by immobilizing glucose oxidase

APPLICATIONS OF CARBON NANOTUBES

207

(Gox) on the surface of CNTs using 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry. The catalytic reduction of H2 O2 produced from enzymatic reaction of Gox upon glucose and oxygen on CNT nanoelectrodes led to selective detection of glucose [8, 36]. 5.5.2 Gene and Drug Delivery Carbon nanotubes of smaller diameter and larger length not only provide higher surface-to-volume ratio for the loading of biomolecules but the inner cavity of nanotubes also provide an excellent carrier for the uptake of biomolecules including DNA and proteins. The facile uptake of biomolecules of varying sizes in the internal cavity of nanotubes and their surface functionalization by adsorption or covalent immobilization has been shown. The functionalized nanotubes can then serve as a vehicle to transport the biological cargo in human cell lines [6]. An alternative strategy has been implied by Ahmed et al. where cationic glycopolymer-functionalized nanotubes were prepared by the covalent immobilization approach, and DNA was complexed on the surface of nanotubes to transport DNA during in vitro studies, as shown in Scheme 5.7. These nanotubes served as an ideal agent for the delivery of nucleic acid with high efficiency [24]. 5.5.3 Tissue Engineering The quest for improved structures of CNTs has focused upon improving their physical properties, including electronic or/and mechanical properties. The CNTs with O

OH O

O

EDC / NHS

O

pH 6, r. t

N

HO O

OH

O

O O

O O

SWNT-COOH

O O

O

O

N

N

O

P(APMA-b-LAEMA)

SWNT-NHS (1) P(APMA-b-LAEMA), pH 9, r. t

(2) pH 6

Sugar groups DNA Amino groups

Complex with DNA

Surface-functionalized SWNT with polymer and DNA

Surface-functionalized SWNT with polymer

SCHEME 5.7 Synthesis of cationic glycopolymer-functionalized SWNTs and their complexation with DNA. (Reproduced with permission from Ref. [24].)

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GLYCOPOLYMER-FUNCTIONALIZED CARBON NANOTUBES

improved properties are then utilized for various purposes including cell culturing. For example, CNTs scaffold functionalized with collagen are found to be suitable for tissue-engineering purposes. The interactions of CNTs with living cells is further based upon the biocompatibility of the former, which is obtained by surface functionalization of these materials with biomolecules [1].

Cellular Interactions The glycosylation patterns on cells are found to play a role in various diseases, for example, tumor-associated alterations on the carbohydrates of cell surface helps their escape from immune response. The characterization of carbohydrates present on the surface of a cell is important to determine their role in various diseases and providing diagnostic tools to treat those diseases. These carbohydrate-based interactions between glycopolymers stabilized nanotubes and pathogenic species such as bacteria or spores can be used to specifically aggregate the pathogenic species in the given medium [13, 37] SWNTs provide a great platform to display multiple copies of bioactive materials, including DNA, proteins, peptides, and carbohydrates due to their high aspect ratio and small diameters, thus enabling effective interactions with living organisms due to exhibition of multivalency on the surface of nanometrials. Bacillus spores are interesting due to their relevant binding to bioactive species and are the focus of nanoresearch due to being a potential bioterrorism threat. It has been found that spore surface is mainly composed of carbohydrates and glycoproteins including galactosamine and 3-O-methyl-rhamnose, thus making them suitable targets for galactose-modified nanoscaffolds. Monosacchridefunctionalized SWNTs have been used to detect and aggregate B. anthracis spores in the presence of divalent cations. Thus carbohydrate functionalized SWNTs have the potential to significantly reduce or eliminate the risk to biological terrorism as aggregated spores are found to be considerably less threatening than the individual ones. Luo et al. [38] have surface-functionalized mannose and galactose residue on the SWNTs and their interaction with Bacillus spores is studied systematically. During this process Ca2+ -mediated interactions of sugar-modified SWNTs was studied in solution, and the extent of colony forming was observed using SEM images and optical microscopy. The extent of colony formation was found to be dependent on the concentration of divalent cations and 100-mM concentration of divalent cations were found to be the optimum for the process. In contrast, no binding was observed upon the incubation of glyconanotubes with spores in the absence of divalent cations. In addition the nature of divalent cations also played a dominant role in colony formation efficiency. For the mannose-f -SWNTs the order of colony formation with respect to the nature of divalent cations is as follows: Mg2+ < Ca2+ < Ba2+ This might be attributed to the diameter of cations, cations of larger diameter being more effective in colony formation and vice versa. It was also concluded that interaction of mannose-functionalized SWNTs with Bacillus spores occurs specifically and is not due to the nonspecific adsorption as Gal-f -SWNTs failed to produce the same response [38].

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27. Jain, K. A., Dubey, V., Mehra, K. N., Lodhi, N., Nahar, M., Mishra, K. D., and Jain, K. N. (2009). Carbohydrate-Conjugated Multiwalled Carbon Nanotubes: Development and Characterization. Nanomed. Nanotech. Biol. Med. 5, 432–442. 28. Shieh, T-Y., and Yang, F-Y. (2006). Significant Improvements in Mechanical Property and Water Stability of Chitosan by Carbon Nanotubes. Eur. Polym. J. 42, 3162–3170. 29. Liu, J., Rinzler, A. G., Dai, H., Hafner, J. H., Bradley, R. K. Boul, P. J., Lu, A., Iverson, T., Shelimov, K., Huffman, C. B., Rodriguez-Macias, F., Shon, Y.-S., Lee, T. R., Colbert, D. T., and Smalley, R. E. (1998). Fullerene Pipes. Science 280, 1253–1255. 30. Ke, G., Guan, W., Tang, C., Guan, W., Zeng, D., and Deng, F. (2007). Covalent Functionalization of Multiwalled Carbon Nanotubes with a Low Molecular Weight Chitosan. Biomacromolecules 8, 322–326. 31. Ke, G. (2010). A Novel Strategy to Functionalize Carbon Nanotubes with Cellulose Acetate Using Triazines as Intermediated Functional Groups. Carbohydr. Polym. 79, 775–782. 32. Dohi, H., Kikuchi, S., Kuwahara, S., Sugai, T., and Sinohara, H. (2006). Synthesis and Spectroscopic Characterization of Single-Wall Carbon Nanotubes Wrapped by Glycoconjugate Polymer with Bioactive Sugars. Chem. Phys. Lett. 428, 98–101. 33. Kitano, H., Tachimoto, K., and Anraku, Y. (2007). Functionalization of Single-Walled Carbon Nanotubes by the Covalent Modification with Polymer Chains. J. Colloid Interface Sci. 306, 28–33. 34. Alpatova, L. A., Shan, W., Babica, P., Upham, L. B., Rogensues, R. A., Masten, J. S., Drown, E., Mohanty, K. A., Alocilja, C. E., and Tarabara, V. E. (2010). Single-Walled Carbon Nanotubes Dispersed in Aqueous Media via Non-Covalent Functionalization: Effect of Dispersant on the Stability, Cytotoxicity, and Epigenetic Toxicity of Nanotube Suspensions. Water Res. 44, 505–520. 35. Worle-Knirsch, M. J., Pulskamp, K., and Krug, F. H. (2006). Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays. Nano Lett. 6, 1261–1268. 36. Kang, X., Mai, Z., Zou, X., Cai, P., and Mo, J. (2007). A Sensitive Nonenzymatic Glucose Sensor in Alkaline Media with a Copper Nanocluster/Multiwall Carbon NanotubeModified Glassy Carbon Electrode. Anal. Biochem. 363, 143–150. 37. Wang, H., Gu, L., Lin, Y., Lu, F., Meziani, J. M., Luo, P. G., Wang, W., Cao, L., and Sun, P-Y. (2006). Unique Aggregation of Anthrax (Bacillus anthracis) Spores by Sugar-Coated Single-Walled Carbon Nanotubes. J. Am. Chem. Soc. 128, 13364–13365. 38. Luo, G. P., Wang, H., Gu, L., Lu, F., Lin, Y., Christensen, A. K. Yang, T-S., and Sun, P-Y. (2009). Selective Interactions of Sugar-Functionalized Single-Walled Carbon Nanotubes with Bacillus Spores. ACS Nano. 3, 3909–3916. 39. Lijima, S. (1999). Helical Microtubules of Graphitic Carbon. Nature 354, 56–58. 40. Valcarcel, M., Cardenas, S., and Simonet, M. B. (2007). Role of Carbon Nanotubes in Analytical Science. Anal. Chem. 79, 4788–4797.

CHAPTER 6

GLYCONANOPARTICLES: NEW NANOMATERIALS FOR BIOLOGICAL APPLICATIONS ISABEL GARC´IA, JUAN GALLO, MARCO MARRADI, and ´ SOLEDAD PENADES Laboratory of Glyconanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE/CIBER-BBN, San Sebastian, Spain

6.1 6.2 6.3 6.4 6.5 6.6

Introduction Gold Glyconanoparticles Gold Glyconanoparticles in Carbohydrate–Carbohydrate Interactions Hybrid Glyconanoparticles Hybrid Glyconanoparticles in Carbohydrate–Protein Interactions Magnetic Glyconanoparticles and Glyco Quantum Dots 6.6.1 Magnetic Glyconanoparticles 6.6.2 Glyco Quantum Dots 6.7 Biomedical Applications: Therapy and Diagnosis 6.8 Future Perspectives and Conclusion Acknowledgments References

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6.1 INTRODUCTION From the three major classes of biomolecules—proteins, nucleic acids—and carbohydrates—the carbohydrates entering into the constitution of glycans and polysaccharides are the least exploited in biology. Their molecular diversity has led to a bewildering variety of species, structures and, characteristics, all performing a large array of functions of great significance. Physiologically, they are useful as Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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FIGURE 6.1 Some examples of carbohydrate-based polymeric particles and gold nanoparticles. (a) SEM micrograph of chitosan particles taken from www.chem.kumamotou.ac.jp/∼ihara/research.html. (b) Glycopolymer (poly-N-isopropylacrylamide (PNIPAAm), methoxy-PEG-SH, biotinylated mannose-polymer) coated gold nanoparticles adapted from Ref. [60]. (c) MannoseC5 S-Au (core radius 0.85 nm) glyconanoparticle modeled by using Sybyl program (Tripos, Inc.) Reproduced with permission from Ref. [5].

energy (nutritional) reserves and for altering the texture and consistency (functional) of foods. The new early advances in glycobiology and glycotechnology have helped to understand the roles of carbohydrates in many biological and physiological processes [1–4], but many aspects must be yet clarified specially at a molecular level. The development of new carbohydrate-based nanomaterials, especially nanoparticles (Fig. 6.1) where functional carbohydrates and novel nanostructures are integrated resulting in new physicochemical properties, opens new avenues for exciting studies on glycobiology and material Sciences. Although carbohydrates play an important part in a vast array of biological processes, they cover only a limited area of the world of therapeutics [6]. Many pathophysiologically important carbohydrate–protein interactions have not yet been exploited as a source of new drug targets. Some reasons might be pharmacokinetic drawbacks of carbohydrates, their low plasma half-life, and the low affinity of carbohydrate-specific interactions in view of targeting cells. Nanotechnology can help to overcome all these limitations by controlling the interactions between carbohydrate-based nanoparticles (from now on glyconanoparticles) and the biological surrounding media. The symbiosis between carbohydrate and materials can provide both with superior properties. Glyconanoparticles (GNPs) can be designed to preserve carbohydrate activity, to enhance their transport of nanomaterials across a biological barrier, and to confer on them a high stability in biological media. At the same time, because carbohydrates are implicated in many cell recognition mechanisms and cell-signaling phenomena, they can be used to increase the targeting specificity toward defined cells in vivo. Multivalent presentation on carbohydratebased nanoparticles allows an efficient targeting by enhancing affinity binding with the corresponding receptor. This review is focused on metallic glyconanoparticles in which the carbohydrate ligands are “covalently” linked to the metallic nucleus and especially devoted to

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highlight examples where the sugars confer biological functionality to the inorganic core of the nanomaterials. The first reports reviewing the work on nanoparticles covered with carbohydrates were published in 2004 and 2006 [7, 8]. Since then, the number of reports on the synthesis, characterization, and application of carbohydrate–nanoparticle hybrid materials has exponentially grown. Glycoconjugates (glycoproteins and glycolipids) are bioactive molecules that play a key role in many normal and pathological processes [9–11]. The mechanisms that govern their behavior depend, at a molecular level, on carbohydrate–protein (carb–prot) interactions [9, 12, 13] and carbohydrate–carbohydrate (carb–carb) interactions [14, 15]. The key characteristic of carbohydrate-based interactions is their extreme low affinity, and the sugars usually need to be arranged in polyvalent clusters with proper orientation and spacing to achieve high affinity. For this reason, a big challenge in current research is the design and synthesis of new polyvalent systems [16] that are able to mimic the natural presentation of biomolecules. Since the pioneer work of Lee on the cluster effect [10], a plethora of multivalent model systems based on peptides, proteins, liposomes, dendrimers, or polymers as scaffolds [17–20] have been prepared to study and evaluate carbohydrate–protein associations. The starting point for the development of glyconanoparticles and glyconanotechnology was our interest to investigate, from a chemical point of view, the existence of carbohydrate–carbohydrate (carb–carb) interactions in water between oligosaccharides, which were proposed as epitopes responsible for cell associations in vivo. In the search for a new polyvalent system, we discovered the potential of the nanotechnology. We developed a new integrated approach based on the use of selfassembled monolayers on two- and three-dimensional surfaces that we have named the glyconanotechnology strategy [7, 8]. A diversity of sugar polyvalent systems based on gold nanoclusters (glyconanoparticles, GNPs) were prepared and applied in the study of carb–carb interactions [7]. The carbohydrate coating, in addition to the antigenic properties, confers stability and solubility in biological media, biocompatibility, and nontoxicity to this glyconanomaterials. In comparison with other multivalent carbohydrate-functionalized systems such as dendrimers [21], polymers [22], and liposomes [23], the glyconanoparticle platform offers some additional advantages. The glyconanoparticle technology allows the preparation of a great variety of water-soluble glycoclusters with different ligand density (high and low loading) and variable linkers to modulate rigidity and flexibility and to confer accessibility to the ligands. The nature (hydrophilic or hydrophobic), the length, and the flexibility of the spacer can be selected to control the presentation of the carbohydrates on the cluster surface, which influences their accessibility to the ligands and behavior during the molecular recognition events.

6.2 GOLD GLYCONANOPARTICLES In general, the methods to synthesize GNPs can be roughly divided into direct (onestep) or multistep approaches. In the one-pot preparation, AuCl4 − salts are reduced with NaBH4 in the presence of the desired thiol-functionalized neoglyconjugate or using noncovalent interaction to introduce the capping ligands [24]. Concerning

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multistep approaches, most of the examples described are based on Brust’s methodology [25, 26] or on Turkevich’s protocols [27, 28]. In addition, a versatile method for the creation of monolayer protected clusters (MPCs) is the place-exchange reaction developed by Murray [29]. In this method glycoconjugate ending thiols displace the native thiols of MPCs in an equilibrium process. Taken together, the control of monolayer structure provided by nanoparticle synthesis, place displacement, and other postsynthetic modification methods can be used to display a wide range of carbohydrate functionality at the particle surface. The first gold glyconanoparticles were obtained in the one-step method by adding a methanolic or aqueous solution of thiol-ending glycoconjugates of the disaccharide lactose (Gal␤1-4Glc␤1) or the trisaccharide Lewis X (Gal␤1-4[Fuc␣1-2]GlcNAc␤1, LeX ) to an aqueous solution of tetrachloroauric acid (HAuCl4 ) and subsequent reduction with NaBH4 [24]. These GNPs were prepared to investigate the selective self-recognition of the LeX antigen via carbohydrate–carbohydrate interaction. This methodology provides a versatile way to prepare in one step a great variety of water-soluble and polyvalent carbohydrate arrays with globular shape. Following this method, the synthesis of GNPs functionalized with different O-glycosides [31] and the tetrasaccharide Ley ([Fuc␣1-2]-Gal␤1-4[Fuc␣1-2]-GlcNAc␤1) [32] has been reported by the same group. The glyconanoparticles prepared in this way are water soluble, stable in solution, noncytotoxic, and the metallic core diameter is a few nanometers (Fig. 6.2a). A similar approach has been taken by the groups of Barchi [33,34], Lin [35,36], and others [37–39]. Kamerling and co-workers prepared glycoAuNP with unprotected thiol-functionalized conjugates, which were obtained by reductive amination [40, 41]. The glycoconjugate obtained by this method can lose its specific binding to proteins, as examined by Liu et al. [42]. Glyconanoparticles have also been prepared by a multistep approach where sugars are conjugated to a previously prepared nanoparticle [43–50]. This method economizes saccharide material but does not ensure control on the degree of functionalization. Otsuka et al. used this procedure in 2001 to introduce lactose and mannose glycosides by reductive amination of an aldehyde-functionalized nanoparticle with the corresponding p-aminophenyl glycosides [43]. Recently, aminooxy-functionalized gold nanoparticles (ao-GNPs) were reacted with natural glycosphingolipids (GSLs) to obtain glyconanoparticles [44]. The synthetic approach involves previous ozonolysis of the double bond in the ceramide moiety of natural GSLs to give an aldehyde and capture them by reaction with the aminoxi-functionalized nanoparticles. The protocol permitted matrix assited laser desorption/ionization-time of flight-mass spectrometry (MALDI–TOFMS)-based high-throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for the adult or GD3 in the case of embryonic mouse. Citrate-capped gold nanoparticles of defined size have been used for the preparation of GNPs by citrate-ligand exchange [45–47]. This approach (Turkevich protocol) permits mild reaction conditions and is highly recommended in the case of biomolecules sensitive to reducing agents. However, the method requires longer reaction time and does not allow for controlled exchange. Manea et al. have recently used dioctylamine-stabilized gold nanoclusters to synthesize GNPs by ligand exchange

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FIGURE 6.2 From left to right: (a) schematic structure of (lactoseC5 S)-Au, computer simulation of (mannoseC5 S)-Au (core radius 0.85 nm, 201 gold atoms) and its TEM micrograph: (b) X-ray crystal structure of the (p-MBA)44 Au102 nanoparticle adapted from Ref. [30].

[48]. This protocol was also applied to the preparation of GNPs with synthetic (oligo)saccharides of type A Neisseria meningitides antigens [49]. Huang et al. proposed the use of tetrakis(hydroxymethyl)phosphonium chloride (THPC) instead of sodium citrate to obtain 2.9 nm of water-insoluble nanoparticles (NPs). These NPs are rendered water soluble by ligand exchange with a thiol-ended glycoconjugate of mannose [50]. Noncovalent stabilization is another possibility to capping gold nanoparticles. Natural polysaccharides (chitosan, heparin, arabic gum, etc.) have been used as reducing and stabilizing ligands [51–56]. However, these natural polymers cannot be considered to be carbohydrates for active targeting. A nanocarrier that contains these

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polysaccharides can improve their interactions with the biological environment and confer high stability and excellent pharmacokinetic properties to the nanoparticle. They can also act as bioadhesive agents to support transmucosal delivery of insulin [57]. Singh et al. produced sophorolipid capped silver NPs mixing a silver salt with the glycolipid in water under basic conditions [58]. By this methodology, biologically active multivalent GNPs incorporating galactose-grafted polymers, obtained by reversible addition–fragmentation chain transfer (RAFT) polymerization, were prepared [59]. GNPs of 40–80 nm were also synthesized using saccharidegrafted poly(N-isopropylacrylamide) (PNIPAM) polymers [60]. In this work, Housni et al. used mannose-grafted glycopolymers and varying amounts of biotinylated polyethyleneglycol, having terminal thiol groups for stabilizing gold nanoparticles. The interaction of the biotinylated GNPs with streptavidin was investigated both by spectrophotometry and surface plasmon resonance (SPR), which confirmed the accessibility and recognition of the biotin moieties. The same authors also reported a novel variant of the synthetic method [60] by in situ photochemical polymerization [61].

6.3 GOLD GLYCONANOPARTICLES IN CARBOHYDRATE–CARBOHYDRATE INTERACTIONS In addition to the well-established protein–protein interaction in cell adhesion, the involvement of specific interactions between carbohydrates in recognition processes was established two decades ago. Part of the glycobiology community focused its interest on this interaction where only carbohydrates are involved, what we refer to as carbohydrate–carbohydrate (carb–carb) interaction. Characteristic features of this interaction is its specificity, its strong dependency on divalent cations, and its extreme low affinity that has to be compensated by multivalent presentation of the ligands. The existence of this interaction is nowadays accepted, but clarification of the mechanisms and further investigation for exploring its implication in other biological processes than the current knowledge is essential for the development of modern glycobiology. Hakomori was the first to describe the implication of a carb–carb interaction in important biological processes as morula compaction and metastasis of lymphoma and melanoma cells in mice [62]. Hakomori studied the expression of different carbohydrate antigens present on GSL domains at the eukaryotic cell surface and their implication in cell adhesion. He proposed a model mechanism where a low-affinity multivalent self-interaction between LeX antigen molecules (LeX -LeX interaction) participated in the initial step of the specific recognition between two cells [63]. This initial step is followed by nonspecific protein–protein interaction that establishes intercellular communication through plasma membrane junction proteins. Another specific GSL–GSL interaction between GM3 and Gg3 or lactose has been proposed as a basis for specific cell recognition between lymphoma and melanoma cells [64]. Several other GSLs involved in carb–carb interactions have also been identified. It has been demonstrated that the interaction between KDNGM3 and Gg3 is involved in the binding of sperm to egg membrane in rainbow trout fertilization [65]. Another example of carb–carb interactions is the specie-specific cell aggregation of marine sponges [66–68]. Misevic et al. [69] demonstrated that an extracellular proteoglycan

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FIGURE 6.3 Carbohydrate–carbohydrate interactions. (a) Glycosphingolipids in specific recognition between cells. (b) Glyconanoparticles as 3D models that mimic GSLs presentation for studying carbohydrate–carbohydrate interactions. (c) Self-aggregation of Microciona prolifera sponge cells mediated by self-interactions of proteoglycan g-200.

containing a sulphated disaccharide (␤-d-GlcpNAc3S-(1-3)-␣-l-Fucp-(1-O)) or a pyruvated trisaccharide (Galp4,6Pyr(␤1-4)GlcpNAc(␤1-3)Fucp) is responsible for the cellular aggregation through a multivalent, homophilic interaction between these carbohydrates (Fig. 6.3). Carb–carb interactions [70, 71] and the study of its mechanism by means of GNPs and biophysical techniques [7] have been already reviewed. A multivalent presentation of carbohydrates, which can allow the analysis of these weak affinity interactions, is essential to study carb–carb interactions. The GNPs nanoplatform mimics the multivalency and reproduces the array of carbohydrates presenting in the patches of GSL clusters at cell surface. The first evidence of the existence of self-interaction forces between the proteoglycan of the Microciona sponge was obtained by means of atomic force microscopy (AFM) [72]. Several multivalent model systems based on vesicles [73], apposed bilayers [74], micelles [75], and artificial glycoviruses [76] have been prepared to study and evaluate carb–carb interactions. de la Fuente and co-workers were pioneers in the design and synthesis of gold nanoparticles decorated with carbohydrates (glyconanoparticles) as multivalent tools to study and intervene in carbohydrate self-interactions [77]. Gold GNPs coated with the trisaccharide Lewis X (LeX ) were used as multivalent models to demonstrate and quantify at the molecular level the calcium-dependent LeX self-aggregation. The antigen determinant LeX seems to mediate morula compaction in mouse via a carbohydrate self-interaction [63]. Conjugates of lactose (used as a control) and LeX having a linear aliphatic (11 carbon atoms) linker were employed for the construction of 2-nm diameter GNPs. The aggregation of lacto- and LeX -GNPs was studied in the presence and absence of calcium cations (10 mM CaCl2 solutions) by transmission electron microscopy (TEM) [77]. LeX -GNPs in calcium solution showed reversible aggregation [the aggregates being dispersible by the addition of ethylene diaminetetraacetic (EDTA) acid] at all the concentrations tested (0.1–0.9 mg/mL) while lacto-GNPs did

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not aggregate under the same conditions. The aggregation is specific so that TEM micrographs showed three-dimensional morula-type aggregates (Fig. 6.4a). Thermodynamic evidence for calcium-mediated self-aggregation of LeX -GNPs was obtained by isothermal titration calorimetry (ITC) [78]. A slow aggregation process with a favorable enthalpy term of about 160 kcal per mole of GNP injected was observed when LeX -GNPs were added to a 10-mM CaCl2 solution. The use of magnesium instead of calcium led to five times lower heat emission, indicating that the LeX aggregation is selectively mediated by Ca2+ (Fig. 6.4b). The heat evolved upon addition of lacto-GNPs to a calcium solution was rather low, the thermal equilibrium was quickly achieved, and the thermal signal confirmed a less strong interparticle interaction. This behavior was not observed in the case of maltose-functionalized GNPs. The selectivity of the aggregation process of LeX -GNPs was also confirmed by SPR [79]. Kinetic data of LeX -LeX interaction were obtained using a combination of self-assembled monolayers (SAMs) of a LeX neoglycoconjugate on a biosensor gold surface as substrate and LeX -GNPs as analyte. The sensorgrams indicated a slow association phase (kon ∼103 M−1 s−1 ) and a gradual dissociation phase (koff ∼10−3 s−1 ) in the presence of calcium cations (Fig. 6.4c). The binding was of high affinity with dissociation constant in the micromolar range (K d ∼10−7 M). The LeX -LeX self-interactions were also confirmed by AFM [80]. The chemical forces measured in calcium solution between self-assembled monolayers of LeX conjugates gave a value of 20 pN for the individual molecules (Fig. 6.4d), while in experiments between self-assembled monolayers of lactose conjugates, no detectable interactions were observed. These results demonstrate the self-recognition capability of this antigen, confirm that carbohydrate self-interactions in water are specific and stabilizing, and establish carb–carb interactions as a mechanism for cell adhesion and recognition. A similar approach has been followed by Carvalho de Souza et al. to explore the carbohydrate-mediated self-recognition of marine sponge cells, which is another example where carb–carb interactions are involved [81]. Ca2+ -dependent self-aggregation of GNPs coated with a sulfated disaccharide epitope (␤-dGlcpNAc3S-(1-3)-␣-l-Fucp-(1-O) (CH2)3 (CH2)6 SH) was observed by TEM imaging [81, 82]. TEM experiments with GNPs coated with the pyruvated trisaccharide (Galp4,6Pyr(␤1-4)GlcpNAc(␤1-3)Fucp (CH2 )6 SH) do not produce visible aggregation [81]. Force curve measurements between tip and gold substrate functionalized with self-assembled monolayers of the sulfated disaccharide showed multiple, stepwise break processes. The correlation function indicates a periodicity composed of multiples of an integer. A force of 30 ± 6 pN was attributed to the interaction of a single disaccharide with itself [83]. In contrast, similar AFM experiments with self-assembled monolayers of the pyruvated trisaccharide did not show any binding in the presence of Ca2+ ions. The same group used NMR spectroscopy [transfer-nuclear Overhauser effect spectroscopy (TR-NOESY)- and DOSY-NMR] to detect the carb–carb self-recognition in solution [84]. When DOSY diffusion experiments and TR-NOESY experiments were carried out in the absence of Ca2+ with a mixture of sulfated disaccharide decorated GNPs and the free disaccharide, no change in the DOSY spectrum and NOE cross-peaks were observed. However, in the presence of Ca2+ ions significant changes in the spectrum and a strong and negative NOE cross-peak were observed, indicating Ca2+ -dependent sugar

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FIGURE 6.4 Evaluating carbohydrate–carbohydrate interactions: LeX -LeX self-recognition is selective and Ca2+ -dependent. (a) TEM micrographs for the interaction between LeX -GNPs. (b) Thermodynamic evidence for self-aggregation of LeX -GNPs as measured by ITC. (c) SPR sensorgrams for the interaction of LeX -GNPs with SAMs of LeX -conjugate. (d) Adhesion forces between LeX -determinant antigens as measured by AFM.

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interactions. A three-dimensional structure for trisaccharide–Ca2+ –trisaccharide complexes obtained by molecular dynamics simulations has been proposed. Reynolds and co-workers prepared GNPs of 16-nm diameter incorporating lactose conjugates with ethylene glycol or aliphatic chains of different lengths [85]. Upon addition of calcium ions the formation of two-dimensional aggregates larger than 100 nm was followed by TEM. A UV–Vis red shift of the surface plasmon absorption band was also observed. The length of the ethylene glycol chain had a remarkable effect on the calcium-induced aggregation. Experiments at different concentration of GNPs have to be carried out in order to confirm the concentration-dependent specific aggregation of these GNPs. Nagahori’s group used aminoxi-functionalized Au nanoparticles (aoGNP)to capture GSL expressed in B16 cells after extraction and ozone treatment of natural mixtures (Fig. 6.5) [44]. This chemical ligation allows capturing between 7 and 16

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FIGURE 6.5 General strategy for structural and functional glycosphingolipidomics based on the selective enrichment of cellular GSLs onto the aoGNP surface by applying the concept of glycoblotting. (Reproduced with permission from Ref. [44].)

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molecules per single nanoparticle of GM3, which is overexpressed in B16 cells. The GM3-functionalized GNPs were used to evaluate, by SPR, the heterotypic interactions between the GNPs capped with GM3 and a Langmuir-blodget membrane of Gg3Cer immobilized on gold sensor chip [44]. This interaction has been proposed as an initial step in the metastasis of melanoma in lung of mice [64]. In this study Nagahori’s group described a protocol that integrates two technologies, “glycoblotting” (glycan enrichment based on chemical reaction) and GNPs-assisted MALDI–TOF, for structural characterization of naturally occurring GSL. The conjugation of mass-limited small molecules onto high-molecular-weight gold clusters, the efficient ionization of thiol compounds chemisorbed onto gold, together with the great ionization efficiency of gold nanoparticles are advantages for their use in MALDI-MS-based structural characterization [86].

6.4 HYBRID GLYCONANOPARTICLES For the study of carb–carb interactions between GSL, a 100% density of carbohydrates on the gold surface is convenient to mimic the GSL patches at the cell surface. However, when other carbohydrate–protein interactions are studied (lectin interactions, enzyme activity), density and the accessibility of the carbohydrates on the nanosurface can play an important role. The glyconanoparticle platform provides the possibility to control the density of sugar residues on the surface and the nature of the linker (“nonspecific interactions”) to obtain the most appropriated model. In addition, the technology allows modifying carbohydrate antigen density, attaching other molecules on the surface of the same nanoparticles, [fluorescent probes, peptides, other cell targets, DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or paramagnetic metal chelates) to get multifunctional nanoplatforms (Fig. 6.6). All components must work in a cooperative way to improve the function of the nanoparticle and allow its detection by different methods. Multifunctional GNPs reported by Ojeda et al. [87] are currently the most complex biofunctional nanoclusters. They described the preparation and characterization of GNPs capped with two different tumor-associated carbohydrate antigens (sialyl-Tn and Lewisy determinants), an immunogenic peptide (a tetanus toxoid TT peptide), and glucose as an inert component to control ligand density (Fig. 6.6b). This platform was designed as a potential carbohydrate-based anticancer vaccine where carbohydrates overexpressed on tumors were conjugated to nanoparticles as the carrier that includes T-cell helper peptides. The “one-pot” procedure allows the controlled assembly of a set of different ligands. Glyconanoparticle technology (glyconanotechnology) allows the preparation of a great variety of water-soluble hybrid glycoclusters with different carbohydrate densities (high and low) in a simultaneous, single, and controlled way. Hybrid lactoGNPs [88], GNPs incorporating glucose and small interfering RNA (siRNA) for transfection and silencing therapy [89], and a small library of (oligo)mannoside GNPs to block HIV (human immunodeficiency virus) gp120 binding [90] are examples of hybrid nanoclusters (Fig. 6.7).

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HO O OH HO OH OH HO OH OH OH HO HO NHAc HO OH OHHO HO OH HO O O OH HOOCO OH HO OH OH O OH OH HO O OH AcHN OH COOH HO OH HO O O O OH O OH O O HOOC NHAc HO O OH O O OH O O OH OHO HO OH O HO HO OH O O OH COOH O O HO O S S OH AcHN O O O AcHN S HO HOOC OH AcHN O S S OH O O O O S NHAc AcHN OH COOHHO S S O OH O HO HO S OH O AcHN O COOH S O O AcHN S OHOH AcHN O S OH HO O OH O S AcHN O NHAc S OH HO S O HV AcHN KW SD LE AV S NS WV S AKIIGIDGLTT HO S O O S HV S S SDKW S SS OH E L V OH OH S VA W S N O O HO LTT V O GIDG WH OH O OH AKII O K O HO ESD O O OH OH OH O OH AVL O O O HO WV HO HO OH OH O OH O NS OH OH HO LTT G OH OH ID HO IIG HO OH AK HO OH HO OH OH OH

Au

(b)

FIGURE 6.6 (a) Schematic representation of hybrid gold GNPs incorporating a diversity of ligands (carbohydrate antigens, peptides, fluorescence dye, RNA/DNA, paramagnetic ions chelating ligands, etc.) with linkers differing in length. (b) Multifunctional GNPs incorporating immunogenic peptides and different carbohydrate antigens. (Reproduced with permission from Ref. [87].)

HYBRID GLYCONANOPARTICLES

O O

O

S SS S S S S S SS O O

O O O

O

O

O O

O

O

O O

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(c)

FIGURE 6.6 (Continued) (c) GNPs functionalized with small interfering RNAs (siRNA) and glucose prepared for transfection and silencing.

A set of hybrid lacto-GNPs have been prepared (Figs. 6.7a and 6.7b), and their interaction with agglutinin from Viscum album and their hydrolysis with ␤galactosidase from Escherichia coli) has been evaluated. Principal conclusions of this work are, first, that lower carbohydrate densities and flexible linkers facilitate the protein recognition and, second, those carbohydrates as 3D multivalent display on the gold surface are resistant to enzyme hydrolysis. [88] A small library of hybrid nanoparticles presenting truncated structures of the undeca-oligosaccharide Man9 (GlcNAc)2 of gp120 (Fig. 6.7c) were synthesized to mimic the cluster presentation of oligomannosides on the HIV envelope protein gp120 and to study their interaction with the dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor expressed on dendritic cells (Fig. 6.7d). The mannose (oligo)saccharides were functionalized with long amphiphilic linkers by different synthetic strategies (direct glycosylation, peptide coupling, or thiourea coupling) to ensure a proper presentation of the antigens, while a glucose conjugate with a short aliphatic chain was selected as an inner stealth component. The synthesis of hybrid glyconanoparticles by ligand exchange is determined by the relative kinetic exchange of the ligands and the nature of the predecessor gold nanocluster different in contrast to the one pot method. Consequently, there is no control over the final ligand ratio. As an example of this procedure, the preparation of hybrid polyethylene glycol GNPs (20 nm) [91], mannose and galactose GNPs have been described [92]. Manea et al. have shown the self-assembly of a thiolated monomeric unit of type A N. meningitides antigens and an inert linker on the surface of gold nanoparticles [93]. According to this protocol, dioctylamine-stabilized nanoparticles were exchanged with mixtures of thiols, resulting in gold nanoparticles that conserve the initial composition of the mixtures. Also, a three-step procedure was used by Zhang and co-workers [94] to prepare hybrid silver glyconanoparticles. Nanoclusters capped with boronic acids, prepared by ligand exchange of tioproninprotected nanoparticles with thiolate boronic acids, were coupled to polysaccharides (dextran 3000) or monosaccharides (glucose). The advantage of this procedure is the low consumption of valuable synthetic carbohydrates.

226

FIGURE 6.7 (a) Lacto-GNPs and (b) hybrid lacto-GNPs for enzyme and lectin interaction. (c) Structure of the high-mannose glycans. (d) Schematic representation of hybrid gold manno-glyconanoparticles. (Adapted from Refs. [88] and [90].)

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6.5 HYBRID GLYCONANOPARTICLES IN CARBOHYDRATE–PROTEIN INTERACTIONS Multivalent carbohydrate–protein interactions (carb–prot interactions) mediate many important physiological and pathophysiological processes. These processes include cellular growth, adhesion, bacterial and viral infections, cancer metastasis, inflammation, and immune surveillance [95, 96] (Fig. 6.8). In nature, carbohydrates at the cell membrane (glycolipids and glycoproteins) are displayed in multivalent presentations. In this way, the weak binding strength of monovalent carbohydrate–protein interactions is compensated with multiple binding events (multivalency). Because carbohydrate–protein interactions play important roles in physiological events, the understanding of their mechanism could provide new therapeutics pharmaceutical leads [97]. A variety of multivalent carbohydrate-based materials have been synthesized to characterize and manipulate these interactions [98–100]. GNPs are one of these carbohydrate multivalent materials that have been shown to be useful tools in this field. In fact, an increased knowledge of these types of interactions will lead to new generations of carbohydrate-based materials. Most of the applications of GNPs in the study of carb–prot interactions have been in the field of biosensing. Different sensing detections and systems have been used. The most popular detection is the colorimetric sensing based on changes in the surface plasmon resonance (SPR) of Au nanoparticles. Apart from colorimetric detection, analytical methodologies based on the different properties of nanoparticles (fluorescence, electrochemical properties) are the more popular techniques used. Otsuka and co-workers were the first to use gold glyconanoparticles for studying carb–prot interactions [43]. They evaluated the reversible interaction between lactoGNPs with bivalent galactose-binding lectin Ricinus communis (agglutinin, RCA120 ) following the changes on the surface plasmon band in the UV–Vis spectrum as a function of time and lectin concentration. A critical lactose density was a requirement for the RCA120 /lacto-GNPs system to induce optically observed particle aggregation. Very recently, the same group employed these GNPs in a colloidal Au replacement assay for the quantitative determination by (SPR) assay of low-molecular-weight analytes (galactose, in this case) [101]. Briefly, preadsorbed lacto-GNPs on the agglutinin (RCA120 ) functionalized sensor chip were eluted by injection of galactose in a concentration-dependent way. The method works fine with a wide range of galactose concentrations (0.1–50 ppm) and the operating time is very short (5 min). Gold nanoparticles enhanced SPR sensitivity and increased the apparent mass of the ligands supported on the nanoparticles. However, it should be stressed that when using GNPs in SPR detection, it is crucial to take into account the nonspecific interactions of gold with the biosensor surfaces. Lectins and more concretely Concanavalin A (ConA) have been the analytes most broadly used as proof of principle for carbohydrate–protein interaction of many carbohydrate-based nanostructures. This plant lectin has been taken as a model to study multivalent effects of different mannose-GNPs. Depending on the methods or detection system, the range of protein detected can be decreased to obtain the

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Virus Cell adhesion

Glycoprotein half-life

Antibodies

Bacterial toxin

Bacteria (a) Types of N-glycans

α2 α2

α2 α3

α3

α2 α6

α6

α6

α6

α6

β4

β4

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β2

β2

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α6

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β

β

Asn

Asn

Oligomannose

Complex

Mannose

Galactose

Fucose

α6

Asn Hybrid N-acetyl-glucosamine

N-acetyl-neuraminic acid (b)

FIGURE 6.8 (a) Schematic drawing illustrating protein–carbohydrate interactions at the cell surface mediating cell–cell binding, cell–microbe (bacterial, viral, and bacterial toxin) adhesion and cell–antibody binding. (Reproduced with permission from Ref. [96].) (b) Types of N-glycans in mature glycoproteins: oligomannose, complex, and hybrid. Each N-glycan contains the common core Man3 GlcNAc2 Asn. (Reproduced with permission from Ref. [100].)

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maximum analytical sensitivity. Lin et al. [102] have demonstrated that nanoparticles are a good multivalent carrier for detecting and quantifying by SPR analysis the interaction between mannose-GNPs and Con A. Aggregation assays with Con A and (oligo)mannose-coated nanoparticles have been described by Hone et al. [103], Karamanska et al. [104], and Halkes et al. [105]. A competitive colorimetric assay for the indirect detection of protein–protein interactions was established based on the Con A/mannose-GNPs (30 nm) system [106]. More interestingly, a biosensor based on microgravimetric quartz crystal microbalance (QCM) has recently been developed in a sandwich-type experiment using mannose-stabilized GNPs as a signal amplifier [107]. Mannose-stabilized gold nanoparticles formed in a sandwich-type complex with the target Con A specifically bound to a mannose-modified Au QCM electrode to give an amplified frequency response. The GNPs prepared by Thygesen and co-workers [47] were also used in Con A aggregation assays. The most interesting result of this work is related to the lower enzymatic stability toward glucoamylase observed for glycan captured by oxime-GNPs compared to glycans capture by oxy-amine-GNPs. These results are due to the lower ligand density of oxime GNPs that could allow more accessibility of the sugars to the enzyme. Similar results were previously described in the case of the hydrolysis of the lactose moiety of lacto-GNP by the ␤-galactosidase enzyme [88]. Modulating the density of lactose conjugates on the gold surfaces, different rates of enzymatic hydrolysis were obtained. Ban et al., described recently a simple method for testing the activity of polysaccharide-degrading enzymes, which are related to many pathophysiological alterations in human diseases, using polysaccharide-GNPs [108]. The method is based on the red shift in the plasmon resonance peak of glycosaminoglycan-protected GNPs caused by the aggregation when these polysaccharide chains are cleaved by the enzyme. Degradation by enzymes (glycosidase, mannosidase, etc.) is an important factor to take into account when designing GNPs as carriers for drug delivery in vivo, and more studies must be done in this direction. Recognition studies of other lectins by nonmetallic GNPs have also been described. The qualitative interaction of mannose biodegradable nanoparticles with recombinant BclA (a dimeric lectin from Burkholderia cenocepacia) was evaluated by a modified enzyme-linked lectin assay (ELLA) and isothermal titration calorimetry (ITC) [109]. Isothermal data provide some insights about the mechanism of the interaction and the contribution of nonspecific adsorption of the lectin in the total recognition event. La Belle et al. described a new electrochemical method for the determination of the amount of glycoproteins produced by to carbohydrate–lectin specific interactions [110]. GNPs conjugated to Thomsen–Friedenreich disaccharide (TF-GNPs), and the glycoproteins asialofetuin (ASF) and fetuin (FET) were run onto a peanut agglutinin (PNA) and Sambucus nigra agglutinin (SNA) immobilized on electrodes of a Cu/Ni/Au printed circuit board (PCB) (Fig. 6.9). TF-GNPs are rapidly detected up to limits of 13 fM on PNA and yielded no response on the SNA electrode. ASF and FET glycoproteins were detectable up to 150 fM on PNA and SNA electrode chips, respectively.

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OH OH

OH O

OH O

O

NH

HO OH

O

O

O

Au

S

6

n

(a) Targets

Targets

PCB electrodes PNA

PNA

SNA

PNA

SNA

immobilized

immobilized

asialofetuin fetuin

TF AuNP

MHDA

SNA

Gold Nickel Copper Fiberglass

(b)

FIGURE 6.9 (a) Structure of Thomsen–Friedenreich antigen–gold glyconanoparticles (TFGNPs) with n = 90–100. (b) Schematic representation of TF-GNP, asialofetuin, and fetuin binding experiments. (Adapted with permission from Ref. [110].)

High sensitivity in protein detection, especially if proteins are biomarkers for diseases, is an important issue. Preparation of florescence nanoprobes is a remarkable way to lower the detection limit. GNPs can be converted in fluorescent probes either by introducing on the gold core surface a fluorescent dye or by modifying the nature of the core. Semiconductor nanocrystals have been used to prepare fluorescent multivalent carbohydrate-coated nanoparticles [glyco-QDs (quantum dots)]. The sensitivity in the detection of proteins was improved by the fluorescence properties of silica-coated lacto- and maltotriose-CdSe-ZnS [111]. Babu and co-workers [112] employed glyco-QDs functionalized with melibiose (d-Gal-␣(1→6)-d-Glc) and lactose (d-Gal-␤(1→4)-d-Glc) in agglutination assays with different lectins. The ␣-galactose-specific agglutination of soybean agglutinin (SBA) was determined by monitoring the binding of melibiose-QD as compared to lacto-QD following scattered light at 600 nm. Multivalent interaction is necessary for agglutination, ␣galactose monosaccharide being ineffective. Maltotriose-QDs were tested with Con A and the results show that concentrations of 100 nM in lectin were still detectable. The preparation and application of glyco-QD will be commented on more extensively in the next section.

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6.6 MAGNETIC GLYCONANOPARTICLES AND GLYCO QUANTUM DOTS 6.6.1 Magnetic Glyconanoparticles Magnetic nanoparticles (MNPs) are a class of nanoscale materials with the potential to revolutionize current clinical diagnostic and therapeutic techniques due to their unique physical properties and ability to function at the cellular and molecular level of biological interactions. Although early research in the field of magnetic nanoparticles dates back several decades, the recent interest in nanotechnology has significantly expanded the breadth and depth of MNP research. Colloidal iron oxide nanoparticles, such as SPIO (superparamagnetic iron oxides) and USPIO (ultra-small superparamagnetic iron oxides), have been the most extensively investigated. Over the course of the past two decades, numerous nanoparticulate iron oxide preparations have been reported and used as tools for cellular therapy, tissue repair, drug delivery, hyperthermia [113, 114], magnetic resonance imaging (MRI) [115–117], magnetic resonance spectroscopy [118], magnetic separation [119, 120], and more recently as sensors for metabolites and other biomolecules [121–123]. Shen and co-workers have developed various formulations of dextran-coated iron oxide nanoparticles also referred to as monocrystalline iron oxide nanoparticles (MION) [124] and crosslinked iron oxide nanoparticles (CLIO) [125], which have been evaluated extensively for a variety of MRI applications. Chemical functionality was established by treating CLIO with ammonia to provide primary amino groups for further conjugation to biomolecules such as proteins [126], peptides [127], small molecules [128], oligonucleotide sequences [129], and optical dyes [130]. Dextran-coated nanoparticles cannot be considered properly glyco-nanoparticles because, in this case, the carbohydrates play a stabilizing role and not an antigenic role. But these nanoparticles give information about the excellence of carbohydrates as biocompatible ligands. They evade the uptake by the reticuloendothelial system (RES) to maintain the stability of nanoparticles and have a long plasma half-life, which are important properties for MNP applications in medicine. In this review we will focus on magnetic glyconanoparticles as probes for targeting carbohydrate receptors. While MNPs conjugated to peptides, proteins, and antibodies have been broadly developed and tested as biological markers, only a few examples of MNPs conjugated to biologicalles relevant carbohydrates have appeared in the literature. We discuss below, in detail, the cases of these magnetic glyconanoparticles (MGNPs). The first water-soluble superparamagnetic gold–iron glyconanoparticles (Fig. 6.10a) covalently functionalized with glucose, maltose, and lactose with a different gold–iron ratio were prepared by reduction in water of a mixture of gold and iron salts in the presence of thiol-ending neoglycoconjugates [131]. The new magnetic GNPs were fully characterized by TEM and ICP-OES (inductively coupled plasma-optic emission spectroscopy), and their magnetic properties were assessed by a superconducting quantum interference device (SQUID). A remarkable result was obtained when comparing the magnetic properties of the Au/Fe containing GNPs and

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(a)

(b)

FIGURE 6.10 Strategies for converting gold glyconanoparticles into superparamagnetic and paramagnetic glyconanoparticles.

pure gold glyconanoparticles. A permanent magnetism to room temperature was observed in the iron-free gold GNPs that was not present in the corresponding gold–iron oxide GNPs [132]. Pure gold nanoparticles with small size (< 2 nm) present magnetic properties. The small particle size and the change on the electronic structure of the nanoparticles due to the covalent gold–sulfur bond seem to be responsible of their ferromagnetism. This behavior was confirmed by the introduction of Fe in a controlled way to gold nanoparticles. Magnetic impurities reduce the high local anisotropic field responsible for the ferromagnetic behavior in thiol-capped gold GNPs [133]. The discovery of magnetic properties on traditionally nonmagnetic materials when in the nanoscale has opened a wide field of research that nowadays is being followed by numerous groups. The interaction of these Fe/Au glyconanoparticles with human fibroblast cell line was investigated using fluorescent and scanning electron microscopies [134]. No cytotoxicity was observed at 20-␮M concentration of glucose and lactose-GNPs, while the maltose-GNPs showed cytotoxicity at 5-␮M concentration. Different cellular responses were obtained for each type of GNP, demonstrating that the cells recognize selectively the sugars on the nanoparticle surface. Another approach to convert directly noble-metal GNPs into paramagnetic ones modifies the organic shell by introduction of a gadolinium complex as an additional ligand (Fig. 6.10b). Hybrid glyconanoparticles, having onto the same gold nanoplatform, sugar conjugates and Gd(III) chelates for converting GNPs into new paramagnetic probes for MRI have been prepared by the one-step synthesis [135]. In the field of contrast agents design, there is also a growing attention toward paramagnetic nanoparticles where Gd(III) ions are incorporated into various nanostructured materials [136, 137]. Thiol-ending neoglycoconjugates of glucose, galactose, or lactose

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FIGURE 6.11 Paramagnetic Gd-based gold glyconanoparticles. (Adapted from Ref. [135].)

were used in tandem with an N-alkyl (pentyl or undecanyl) tetraazacyclododecane triacetic acid (DO3A) derivative to coat the gold nanoclusters (Fig. 6.11). The longitudinal (T 1 ) and transversal (T 2 ) relaxation times were determined, and the relaxivity (r1 ) values were calculated from T 1 [the slope of 1/T as a function of millimolar Gd(III) concentration] and compared with in clinical use of Dotarem. Both sugar stereochemistry and the relative position (l) of the sugar with respect to the Gd(III) ion seem to control the relaxivity values of these GNPs. The best GNPs yielded relaxivities above 20 mM−1 s−1 , which is over six times the values of Dotarem. These GNPs were used for in vivo imaging of glioma in mice. To the best of our knowledge, there are no other examples of GNPs that incorporate Gd(III) in the organic shell. However, dextran-coated GdPO4 [138] or Gd2 O3 [139] nanoparticles have been reported. Contrary to the one-pot synthesis used for the preparation of the previous T 1 -gold MGNPs, T 2 -based magnetic glyconanoparticles have been synthesized by chemical functionalization of as-prepared iron oxide magnetic cores. The possibility of inserting antigenic carbohydrates with this approach allow maintaining the magnetic performance of the cores but requires the presence of reactive functional groups (amine, caboxylic, alkinyl, azide, etc.) in both the surface of nanoparticles and the glycoconjugates to attach the carbohydrate antigens. The last strategy in which magnetic nanoparticles with good physical properties according to the final application are chemically modified has been chosen by Kasteren et al. [140] for insertion of antigenic carbohydrates. The success of the Kasteren et al. approach is to apply on the nanomaterials the combination of enzymatic and chemical glycosylation that the authors had previously used for engineering glycosylated enzymes. Amine groups present on crosslinked iron oxide dextran-coated nanoparticles were glycosylated by using the 2-imido-2-methoxy-ethyl (IME) method. In this method the IME group was masked as the S-cyanomethyl group because this group is chemically inert to glycan deprotecting conditions. Finally, the last step

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involved the activation of S-cyanomethyl to give IME and consequent coupling to the amine-functionalized MNPs. Multiple GlcNAc, LacNAc, sialyl LacNAc, and sialyl Lewis X (sLeX ) ligands were introduced to exploit needed carbohydrate clustering. The sLeX -GNP prepared to target CD62 (E- and P-selectin) receptors allowed the direct detection of activated endothelial cells in acute inflammation by means of MRI. Silica-coated iron oxide magnetite nanoparticles were used by the El-Boubbou group to conjugate ␣-mannose or ␣-galactose derivatives by covalent functionalization [141]. Tetraethoxysilane was used to build up the first silica shell on the magnetic NPs. Triethoxysilane derivatives endowed either with a terminal alkyne or with an amine group were further used to functionalize the shell. In this way, it was possible to immobilize on the nanoparticle surface suitable carbohydrate derivatives by click chemistry or peptidic coupling. The last two examples of MGNPs will be carefully reviewed later in the last section devoted to biomedical applications. Other approaches based on noncovalent functionalization of the nanoparticle cores with carbohydrates have been reported. Biotinylated bi-antennary or tetraantennary glycoconjugates of sialic acid trisaccharide, ␣-mannoside, ␣-lactoside, and ␣-thiosialioside were used to functionalize avidin-coated magnetic beads [142]. d-Mannose-modified iron oxide nanoparticles were prepared by random absorption and used for stem cell labeling [143]. Also d-gluconic acid, lactobionic acid, and Ficoll protected iron oxide nanoparticles that were synthesized by direct absorption during their synthesis at 75–80◦ C by Kekkonen et al. [144]. The synthesis of stable and aqueous redispersable 50-nm cobalt superparamagnetic nanoparticles protected with sophorolipids (a class of glycolipids obtained by incubation of glucose and fatty acids with yeasts) as a water-soluble capping agent was achieved by NaBH4 reduction of cobalt chloride [145]. The saturation magnetization value of this cobalt GNPs (23 emu/g) was higher than that of simple oleic-acid-capped cobalt nanoparticles (3 emu/g) prepared under similar conditions, although differences in size, chemical composition, and capping nature may play a key role in tuning the magnetic properties. Nowadays an intensive research is focused on providing multimodal magnetic nanoparticles where the biomolecule plays a key role both as targeting moiety and as biocompatibility agent. The success of the application of these multimodal MNPs in diagnosis and disease treatment will depend on the understanding of the relationships between their physicochemical properties (cluster and shell ligands) and their behavior in vivo. The present knowledge is insufficient to predict biodistribution, toxicity, or in vivo behavior and a more exhaustive investigation is necessary in this direction. 6.6.2 Glyco Quantum Dots Fluorescent semiconductor nanocrystals (CdSe, CdTe, InP, etc.), otherwise included in the term quantum dots (QDs) [146], have attracted much attention in different research fields for more than 20 years [147, 148] because of their chemical and physical properties, which markedly differ from those of the bulk solid (quantum size effect) [149, 150]. Quantum dots have size and matter-tunable light emission (usually with a narrow emission band), bright luminescence (high quantum yield), long emission stability (photobleaching resistance), and broad absorption spectra

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235

(b)

(a) (c)

FIGURE 6.12 (a) Absorption and emission of six different QD dispersions. The black line shows the absorption of 510-nm emitting QDs. (b) Representative QD core materials scaled as a function of their emission wavelength superimposed over the spectrum. Representative areas of biological interest are also presented corresponding to the pertinent emission highlighting how most biological usage falls in the visible–near-infrared region. (c) Photo demonstrating the size-tunable fluorescence properties and spectral range of the six QD dispersions plotted in (a) versus CdSe core size. All samples were excited at 365 nm with a UV source. (Reprinted with permission from Ref. [162].)

for simultaneous excitation of multiple fluorescence colors compared with classical organic fluorescent dyes (Fig. 6.12). This last property has been used to excite a dual construct formed by QDs conjugated with fluorescent-labeled poly(lactideco-glycolide) (PLGA) nanoparticles and to image cells at two different emission wavelengths by using the same energy [151]. Research on QDs has evolved from electronic materials science to biological applications [152–156]. There are numerous reports on synthetic methods for the preparation of QDs, their conjugation with biomolecules, and their application as bioluminescent probes for live-cell and/or in vivo animal imaging [157–165]. Since 1982 Efros [166] and Ekimov and Onushchenko [167] built semiconductor nanoand microcrystals in glass matrices, and later in 1993 Murray et al. [168] described the synthesis at high temperatures of high-quality colloidal CdS, CdSe, and CdTe quantum dots with narrow size distributions. Core@Shell CdSe@ZnS [169, 170] has become the most common chemical composition for QDs synthesis, especially for biological applications. Synthesized hydrophobic QDs cannot be directly used in bioapplications due to their insolubility in water. Substitution/modification/encapsulation with the capping molecules to render water-soluble QDs results in considerably decreased photoluminescence [171–173]. This is the main reason why in the last few years direct aqueous synthetic methodologies are gaining attention. Nowadays protocols developed to prepare water-soluble CdTe quantum dots in the presence of thiol molecules as capping agents have provided QDs with high quantum yields [174–176].

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FIGURE 6.13 Main strategies for the synthesis of glyco-QDs by means of thiol chemistry.

Water-soluble glyco quantum dots (glyco-QDs) have been prepared following two different strategies similar to those for magnetic glyconanoparticles, either directly by in situ reaction with derivatized carbohydrates or in several steps that comprise the modification of as-synthesized luminescent cores (Fig. 6.13). In this last strategy, carbohydrates have been capped onto QD surfaces by means of thiol chemistry or noncovalent interactions (electrostatic, hydrophobic, or biological forces). Quantum dots conjugated to carbohydrate antigens for specific cell targeting have been prepared by the direct methodology using aqueous solutions of thiolfunctionalized neoglycoconjugates and metal salts. The preparation of cadmium sulfide (CdS) nanocrystals covalently bound to the antigenic trisaccharide LeX or to maltose derivatives was accomplished in a single step by adding, at room temperature and pH = 10, sodium sulfide to a water solution of thiol-ending neoglycoconjugates and cadmium nitrate [177]. The obtained QDs were water soluble, stable (in the absence of light at 4◦ C), and emitted light at 550 nm when excited. This type of aqueous self-assembly procedure has been also used for the generation of mannose-conjugated CdS quantum dots [178]. QDs coated with the tumor-associated Thomsen–Friedenreich antigen conjugated to various linkers were also one-pot synthesized by the use of a small percentage of different surface passivating agents and suitable neoglycoconjugates [179]. Binding and agglutination assays confirmed that the functional characteristics of the sugar were intact on the particles. Two-step protocols have been also used to prepare glyco-QDs. They consist in preparing QDs with classic capping agents, which are then subjected to ligand

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exchange with suitably functionalized thiol-ended glycoconjugates. Pyridineencapsulated QDs were treated with 11-mercaptoundecyl ␤-N-acetylglucosamine (in its disulfide form) and NaBH4 in aqueous solution to give the water-soluble ␤-N-acetylglucosamine-QDs [180]. Sulfur atoms mediate the linkage to colloidal CdSe@ZnS core@shell QDs by displacement of pyridine. Very recently, a thiolending triantennary dendritic ␤-galactoside ligand was anchored to pyridine-capped CdSe@ZnS nanoparticles by a similar procedure [181]. Likewise, CdSe@ZnS QDs with lactose, melibiose, and maltotriose on their surface have been synthesized by thiol coupling of the thiol-derivatized sugars with the ZnS shell using tetramethylammonium hydroxide as initiator [112]. This same two-step methodology has been used to synthesize N-acetyl glucosamine, glucose, galactose, and mannose displaying CdTe quantum dots by surface exchange in water of mercapto-propionic acid (MPA) by thiol ending neoglycoconjugates [182]. By using the same protocol, oligosaccharides (maltose, maltotriose, cellotriose, and mannose) were also incorporated into CdTe quantum dots [183]. The synthesis of glyco quantum dots has also been achieved by multistep protocols starting from readily prepared QDs that are not soluble in water. Replacement of the hydrophobic ligands by water-soluble bifunctional molecules in which one terminus (usually a thiol) is used to anchor to the surface of QD, and the other end is a reactive functionality (like amine, carboxylic acid, etc.) allows the coupling of suitable glycoconjugates (Fig. 6.13). Trioctylphosphine oxide (TOPO)-capped InGaP@ZnS QDs were exchanged with mercapto-hexadecanoic acid and then activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to covalently couple deacetylated (85%) chitosan [184]. CdSe@ZnS nanocrystals coated by amino-functionalized silica were conjugated with activated dextran to prepare watersoluble and stable QDs [111]. CdSe@ZnS QDs capped with galactose, mannose, and galactosamine have also been prepared in three steps. In this work, ligand exchange of TOPO-capped QDs with thioctic acid coupled with diamino-PEG2000 in reducing conditions afforded the intermediate QD-PEG2000 -NH2 . The terminal amine was further reacted with the carboxylic group of a spacer endowed with a maleimide. In this way, carbohydrate-linked thiols were inserted by maleimide conjugation [185]. Nowadays QDs with good luminescent and biocompatibility properties are already commercially available, and some groups have chosen to modify these kinds of QDs instead of synthesizing them. In these cases the emission wavelengths in the market are limited, and information on the concentration and functionalization of the nanocrystals is scarce. High biocompatible mannosylated QDs were prepared starting from commercially available amino-polyethyleneglycol-coated QDs (Qdot 655 ITK incorporating polyethyleneglycol PEG2000 with M w -2000) [186]. Also commercially available QDs containing carboxylic groups at their surface were reacted with biotinylated glycopolymer containing amino functionalities or with the mixture of amine-functionalized biotin and N-(2-aminoethyl)gluconamide [187]. In this work, the pyranose ring of glucose was disrupted by chemical conjugation with the linkers. Coupling of commercially available streptavidin-coated QDs with a biotinending lactose-grafted glycopolymer was achieved at room temperature and short times (1 h) to obtain fluorescent bioprobes [188].

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gp 120-QD

Concerning methods based on noncovalent interactions, the first reported synthesis of glyco-QDs followed this strategy by mixing negatively charged carboxy-methyldextran and mercaptosuccinate-capped CdSe@ZnS QDs with positively charged polylysine [189]. This noncovalent strategy has been used to prepare chitosan-coated QDs [53, 190–194], carboxymethyl chitosan QDs [195], calix[4]arene-based glycoclusters (␣- and ␤-glucoside, ␤-galactoside, and ␣maltohexaoside) [196], and gum arabic protected QDs [197]. Recently, biotinylated polymeric LeX or HIV-1 envelope glycoprotein gp120 were bound to commercially available PEG-coated fluorescent QDs (Qdot 605 ITK) conjugated with streptavidin to obtain ∼40 nm overall diameter systems [198]. In this interesting work, the glyco-QDs were applied for targeting DC-SIGN in cellular models. Binding and internalization of virus-sized (40-nm) QD conjugates by chinese hamster ovary (CHO) cells that express human DC-SIGN and dendritic cells (DCs) was visualized by confocal microscopy. Imaging demonstrated that both pLeX and gp120-QDs were internalized by DC-SIGN-mediated processes. The mechanism of this internalization was found to be clathrin dependent and caveolin independent. In the same work, the authors investigated the fate of gp120-QDs conjugates once uptaken by the cells. Fluorescence colocalization into the lysosomal compartment first, and into the MHCII (major histocompatibility complex class II) enriched compartment later, was observed (Fig. 6.14). This result suggests that the conjugation of antigens to QDs alters neither cell viability nor the proper antigen degradation inside lysosomes and subsequent loading of MHCII molecules. Niikura and co-workers studied the uptake of different monosaccharidedisplaying (N-acetylglucosamine, glucose, galactose, mannose) CdTe quantum dots by digitonin-permeabilized HeLa cells [182]. They demonstrated that only GlcNAcfunctionalized QDs are taken up by the cells, and that this interaction is adenosine 5 triphosphate (ATP) dependent. They propose a specific interaction between GlcNAc and the protein GRP78/Bip of the HSP70 family present in the endoplasmatic reticule (ER). This result highlights the potentiality of glyco-QDs as tools for the study of cytoplasm glycobiology, which is by far the less known one. The same group has recently reported that QDs coated with oligoglucosides (maltose, maltotriose, cellotriosa, panose) are transported into the nucleus of digitonin-permeabilized HeLa cells without the use of cationic nuclear localization signals [183]. Mono-glucopyranosidedisplaying QDs (irrespective of the anomeric configuration of the glucoside) were

HLA-DR

FIGURE 6.14 From left to right: Single optical section of dendritic cells labeled with antiHLA-DR mAb (green), after 90-min incubation with gp120-QD605 (red) and the colocalization image. Right panel: 2D histograms of HLA-DR signal vs. gp120-QD605 signal. Scale bars represent 10 ␮m. (Adapted from Ref. [197].)

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MAGNETIC GLYCONANOPARTICLES AND GLYCO QUANTUM DOTS

O

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OH O

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OH O OH O

OH O HO

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H O O

O

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C

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H

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s no

se

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Pa

0

al

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SH HO

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(a)

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1

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OH O

Fluorescent intensity in nucleus

O R

MPA–QD

Sugar–QD

(b)

(c)

FIGURE 6.15 (a) Chemical structures of PEG 1, and neoglycoconjugates ␤-glucose 2, ␣glucose 3, maltose 4, maltotriose 5, panose 6 and cellotriose 7. (b) Synthesis of sugar-displaying QDs by thiol exchange reaction. (c) Confocal fluorescence (top panels) and differential interference contact (DIC; lower panels) images of digitonin-permeabilized HeLa cells incubated with sugar-displaying QDs. (d) Digitalized fluorescence intensity of selected QDs in the nucleus. (Adapted with permission from Ref. [183].)

retained in the cytosol and not translocated into the nucleus. A marked accumulation of maltotriose- and panose-QDs in the nucleus was observed (Fig. 6.15) while only a few maltose-QDs were seen in the nucleus. The fluorescent intensities in the nuclei from the sugar-displaying QDs followed the order trisaccharide > disaccharide >> PEG linker. Glyco-QDs have been used to study endocytosis widely. As examples of receptormediated endocytosis, Robinson’s group used N-acetylglucosamine-conjugated and mannose-encapsulated CdSe/ZnS core–shell QDs to stain sperm lectins by fluorometry [180]. Chen et al. demonstrate that galactose-functionalized QDs entered into HeLa, kidney, and lung cancer cells (Fig. 6.16) by a receptor-mediated endocytosis mechanism [181]. Higuchi and co-workers used mannosylated QDs for the labeling of peritoneal macrophages [186]. Examples of nonspecific internalization have also been found. Osaki and coworkers have used glyco calix-[4]resorcarene-QD conjugates as endosome markers [196]. Xie et al. compared the uptake of CdSe/ZnS-labeled carboxymethyl chitosan against CdSe/ZnS-labeled mercaptoacetic acid by yeast cells. [195] Sandros and

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GLYCONANOPARTICLES: NEW NANOMATERIALS FOR BIOLOGICAL APPLICATIONS

(b)

(a)

(c)

FIGURE 6.16 (a) Scheme of galactose functionalized CdSe@ZnS QDs. Right: Images taken from incubation experiments of A549 lung cancer cells with 10 mM of triantennary, galactosidic nanohybrid-12 for 24 h. (b) A stacked bright-field and fluorescence image. (c) Confocal microscopic image with Z-clipping along x and y axes. (Adapted with permission from Ref. [181].)

co-workers used covalently chitosan-encapsulated InGaP/ZnS quantum dots emitting at 670 nm for deep-tissue imaging [184].

6.7 BIOMEDICAL APPLICATIONS: THERAPY AND DIAGNOSIS Nanotechnology is offering an outstanding contribution to understand biological processes at a molecular level and in the development of diagnostic tools and innovative therapies. The current advances in the engineering of optical imaging techniques have revolutionized both basic research and clinic applications in diagnostics. There is an increasing demand for new probes that can guarantee not only high stability, specificity, and low toxicity but that can also be employed simultaneously in different imaging techniques with high sensitivity. Dye-doped silica nanoparticles, quantum dots, and gold nanoparticles have been prepared and applied for noninvasive bioimaging [199]. Bioconjugated quantum dots are emerging as powerful luminescent probes

BIOMEDICAL APPLICATIONS: THERAPY AND DIAGNOSIS

241

and have been used both for live-cell labeling and in vivo animal imaging [155]. Absorbance and emission in the near-infrared window are expected to allow real-time and deep-tissue optical imaging. In the area of magnetic resonance imaging (MRI), magnetic nanoparticles have demonstrated to be highly sensitive and target specific for observing biological events both at cellular and molecular levels [200, 201]. The development of multimodal nanoparticles that are both optical and MRI active is probably one of the most important advances in applied nanotechnology. A similar development is observed in the application of biocompatible nanomaterials in innovative therapies as gene and drug delivery [202]. Nanomaterials have also been used for controlled release of therapeutic agents, specially focusing on cancer therapy [203]. Recently new nanomaterials have been developed to treat nervous system disorders [204]. Although any immediate clinical application of these new nanomaterials is still far away, some of the GNPs described here have been applied in preclinical studies to approach biomedical problems. Glyconanoparticles constitute a good biomimetic model to intervene in carbohydrate-mediated biological processes. The adhesion of microbes to host cells can occur via carbohydrate–protein and/or via carbohydrate–carbohydrate interactions. The discovery of the important role of carbohydrates in recognition processes has suggested a new antimicrobial therapy based on their antiadhesion potential. The use of antimicrobial adhesion agents based on carbohydrate analogs of host glycoconjugates has been envisaged as a possible alternative to current antibiotic-based treatments [205]. The construction and application of glyconanoparticles with these characteristics is still in the embryonic state. The application of different types of carbohydrate-functionalized nanomaterials in the detection and control of microorganism has been reviewed [206]. The first application of gold GNPs as antiadhesion agents against lung metastasis progression in mice was reported in 2004 [207]. Ex vivo preincubation of tumor cells with lacto-GNPs inhibited up to 70% metastasis of melanoma cells in lung of mice. An ex vivo experiment was designed for the evaluation of the antimetastasis potential of the glyconanoparticles. Mice were injected with melanoma cells preincubated with lactose gold glyconanoparticles, and after 3 weeks the animals were sacrificed and both lungs evaluated under the microscope for analysis of tumor foci. Tumor inhibition was reported as compared with the groups inoculated only with melanoma cells or with melanoma cells incubated with gluco-GNPs (Fig. 6.17). In an attempt to intervene in HIV entry processes, glyconanoparticles have been used to investigate the role of multivalent interactions of HIV envelope glycoprotein gp120 with host cellular receptors. Galactosyl ceramide (GalCer) is a glycosphingolipid (GSL) expressed on mucosal membrane cells, which is implicated in viral entry in these kinds of cells [208]. Nolting et al. [39] investigated the polyvalent interactions of gold galacto- and gluco-GNPs with recombinant gp120 (rgp120). A biotin-neutravidin adhesion assay was employed to evaluate the ability of the GNPs to displace rgp120 from plate-bound GalCer. Divalent disulfides of the tested glycoconjugates were much less active than biotinylated GalCer. However, when these conjugates were multivalently presented on the GNPs, they were two orders of magnitude more active than the disulfides and at least one order of magnitude more active than biotinylated GalCer.

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GLYCONANOPARTICLES: NEW NANOMATERIALS FOR BIOLOGICAL APPLICATIONS Glc-GNP (90 µm)

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FIGURE 6.17 (a) Schematic representation of ex vivo experiments for evaluating the antimetastatic potential of lacto-GNPs. (b) Analysis of the effect of gluco-GNPs and lacto-GNPs on the B16F10-dependent development of lung tumoral foci at two different magnifications (×8 and ×80). Black arrows indicate small foci (<1 mm) and blue arrows indicate large foci (>1 mm). (c) The specific antimetastatic effect of lacto-GNPs. Lungs corresponding to mice treated with B16F10 cells (mock), gluco-GNPs (second row), or lacto-GNPs (third row) in comparison with the lungs obtained from a control animal (not injected with B16F10 cells; top right). (Adapted from Ref. [207].)

´ To construct potential microbicides against HIV-1 infection, Mart´ınez-Avila´ s group has designed and synthesized GNPs coated with oligomannosides of the glycans on gp120 with the aim of mimicking the virus itself. A small library of gold GNPs coated with sets of different structural motifs of the N-linked high-mannose undecasaccharide Man9 (GlcNAc)2 of gp120 were prepared and characterized [90]. Mannose (oligo)saccharides with different spacers and in variable density were inserted on the gold nanoplatform. These manno-GNPs were designed to target DCSIGN (dendritic cell-specific ICAM 3-grabbing nonintegrin) receptors present on dendritic cells (DCs). These cells can transfer the virus to T lymphocytes where viral replication occurs through a mechanism named transinfection [209]. The HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN expressed on DCs [210]. SPR experiments were used to test the inhibition potency of some selected mannosides-containing GNPs toward DC-SIGN binding to immobilized gp120. These GNPs completely inhibited the binding from micro- to nanomolar range, while the corresponding monovalent mannosides do not show any inhibition

243

BIOMEDICAL APPLICATIONS: THERAPY AND DIAGNOSIS

at these concentrations. GNPs containing the disaccharide Man␣1-2Man␣ were the best tested inhibitors showing more than 20,000-fold increased activity (100% inhibition at 115 nM) compared to the corresponding monomeric disaccharide (100% inhibition at 2.2 mM) [90]. These GNPs were able to inhibit the DC-SIGN-mediated HIV transinfection of human-activated peripheral blood mononuclear cells at nanomolar concentrations [211]. Raji cells expressing the receptor DC-SIGN were incubated with GNPs and then pulsed with HIV recombinant viruses (Fig. 6.18). After washing, the cell cultures were co-cultured with human-activated peripheral blood mononuclear cells (PBMCs) that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was assessed by luciferase activity in cell lysates. This experimental Raji-DC-SIGN+

O Glucose

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-D C

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te d re a nt

AJ ID R

U

00

0%

(c)

FIGURE 6.18 (a) Cell-based experiments for evaluating the anti-HIV potential of mannoGNPs as inhibitors of DC-SIGN-mediated HIV-1 transinfection of human T cells. (b) Schematic representation of manno-GNPs. D, T, Te, P, and H stand for di- tri-, tetra-, penta-, and heptamannose conjugates, respectively; the numbers indicate the percentages of mannose oligosaccharides on GNP, the rest being the stealthy GlcC5 S conjugate component. (c) Anti-HIV evaluation of manno-GNPs at 1 mg mL−1 in DC-SIGN-mediated trans-infection of human T cells. HIV-1 recombinant viruses JR-Renilla R5 (striped) or NL4.3-Renilla X4 (black) were used. Raji cells not expressing DC-SIGN (Raji DC-SIGN-) were used as control to allow for DC-SIGN-independent viral transfer. Mannan (100 mg mL−1 ) was used as a positive control. Results are expressed as percentages of infection related to untreated control. (Adapted with permission from [211].)

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GLYCONANOPARTICLES: NEW NANOMATERIALS FOR BIOLOGICAL APPLICATIONS

setting tries to mimic the natural route of virus transmission from DCs to T lymphocytes. In this way, we proved that synthetic carbohydrate-based multivalent systems can prevent viral attachment to DC-SIGN-expressing cells and thus may function as an antiadhesive barrier at an early stage of HIV infection. A prerequisite for many microbial infections (bacteria, toxins, viruses, etc.) is adhesion of the infecting organisms to host cells through multivalent protein– carbohydrate binding. Given the increased incidence of bacterial resistance to traditional antibiotics, the development of carbohydrate-based antiadhesion agents presents a promising approach for the prevention of susceptible microbial infections. In this field, several examples have been described using glyconanoparticles as antiadhesive agents. Mannose-coated magnetite nanoparticles have been employed by El-Boubbou et al. [141] for rapid detection of E. coli (up to a limit of 104 cells/mL) and for the removal of up to 88% of the target bacteria from the medium. Mannose-encapsulated gold nanoparticles have been successfully tested to bind mannose-specific FimH proteins on the type 1 Pili in E. coli [35]. Manea’s group used GNPs endowed with synthetic analogs of the repeating unit of the capsular polysaccharide of type A N. meningitidis to inhibit the binding of this bacterium to a specific mouse polyclonal antibodies of the natural serotype A as determined by enzyme-linked immunosorvent assay (ELISA) assays [93]. Toxins have been already detected with different glyconanoparticles. Recently, globotriose-functionalized GNPs were employed in SPR competition binding assays with the pentameric B-subunit of Shiga-like toxins (B-Slt) that specifically recognizes globotriaosylceramide (the globotriose blood group antigen) [212]. Globotriose-GNPs showed sizeand linker length-dependent affinity for the protein and were used as multivalent probes for the purification of the B subunit from cell lysates. Two relevant samples of the application of glyconanoparticles in molecular and cellular imaging are the works of Lee et al. [213] with near-infrared fluorescent gold glyconanoparticles, and of Kasteren et al. [140] with superparamagnetic glyconanoparticles. The first study presents in vivo imaging of arthritic inflammation and human ovarian carcinoma (OVCAR-3) tumor in mice upon systemic injection with multifunctional gold GNPs capped with hyaluronic acid labeled with a near-infrared fluorescence dye [213]. The fluorescence quenching by energy transfer between the dye and the gold surface is deactivated when the GNPs reach the target zones (arthritic joints and tumors) where reactive oxygen species and hyaluronidase are overexpressed and synergistically degrade hyaluronic acid moieties. The dye release allows the ultra-sensitive detection of these disease states by in vivo fluorescence imaging. These results suggest that gold nanoprobes can be exploited not only as in vitro molecular and cellular imaging sensors, but also as in vivo optical imaging agents for detection of local hyaluronic acid degrading diseases. Finally, MRI-active dextran-coated iron oxide nanoparticles functionalized with sialyl Lewis X (sLeX ) were used for in vivo inflammation detection (Fig. 6.19). The sLeX -GNP constructs were highly sensitive and selective T2 contrast agents for detecting endothelial markers E-/P-selectin (CD62E/CD62P) in acute inflammation. T1 -weighted images with the gadolinium-based contrast agent Omniscan verified a lack of blood–brain barrier breakdown at the end of the GNP protocol. The good

FUTURE PERSPECTIVES AND CONCLUSION

OH OH

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OH HO

OH

OH O

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χ105

GNP-sLex

FIGURE 6.19 Left: Synthesis of iron oxide glyconanoparticles. Right: Selected images taken from the T 2 ∗ -weighted 3D datasets (A, C, and E) and 3D reconstructions of the accumulation of contrast agent (B, D, and F); sLeX -GNP enables clear detection of lesions in clinically relevant models of multiple sclerosis (C and D) and stroke (E and F) in contrast to unfunctionalized control-NP (A and B). (Adapted from Ref. [140].)

correlation observed between the in vitro data for those particles that bind well to E-selectin (sLeX -GNP and sLeX -GNP-FITC), and those that do not (LacNAc-GNP) with the binding observed in vivo suggests that this may be mediated by expressed selectin biomarkers (Fig. 6.19).

6.8 FUTURE PERSPECTIVES AND CONCLUSION Glycans (otherwise called carbohydrates, sugars, saccharides, and oligosaccharides) and their conjugates (glycoproteins, glycolipids, proteoglycans) are abundant and biological significant molecules. In spite of this, their use as biofunctional molecules in nanobiotechnology is behind from their partner proteins and nucleic acids. The broad diversity of their complex structures has impeded the characterization of their biological functions. Information about carbohydrate–protein and carbohydrate–carbohydrate interactions involved in may normal and pathological processes decode the dense structural information contented in glycans and glycoconjugates. New specifities of carbohydrate-binding proteins (lectins) are continuously been elucidated thanks to new technologies such as glyco-microarrays. This technology identifies glycan-binding epitopes, which provide information of carbohydrate-related targets for the design of new therapeutics. Glyconanoparticles represent a unique 3D-platform that has to be added to the 2D-glycoarray technology. The glyconanoparticle platform can help to overcome drawbacks that are

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inherent to carbohydrate as drugs (short half-life in plasma, fast renal excretion, and high polarity) conferring resistance to the oligosaccharides against glycosidases. Reciprocally, carbohydrates confer singular properties to metallic nanoclusters for biomedical applications such as water solubility and biocompatibility. Carbohydrates avoid nonspecific interaction of proteins similarly as polyethylene glycol chains but without producing an immuno-response, help to cross cellular membranes, and control the fate of the nanoparticles inside the cell. The combination of carbohydrate with nanomaterials may improve the efficacy for targeting specific sites. In this review, we have presented the few biomedical applications of glyconanoparticles described until today in antiadhesion therapy in metastasis of melanoma, as inhibitors of the adhesion of virus bacteria and toxins and as probes in fluorescence and magnetic resonance imaging. Considerable work remains to be done on the integration of biologically significant carbohydrates into nanosystems to develop more exciting functions and applications and to answer some relevant incognita. Both carbohydrate-based micro- and nanomaterials can significantly contribute to the development of Glycobiology and Glycomics.

ACKNOWLEDGMENTS The authors thank the many colleagues who have contributed along the years to the development of the glyconanotechnology. Experimental studies described in this report were supported by the Spanish Ministry of Science and Innovation (grant CTQ2008-04638), the European Union (grants EMPRO, LSHP-CT2003-503558, and CHAARM, Health-F3-2009-242135), the Department of Industry of the Basque Government (grant ETORTEK) and CIBER-BBN.

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CHAPTER 7

GLYCODENDRIMERS AND THEIR BIOLOGICAL APPLICATIONS ELIZABETH R. GILLIES Department of Chemistry and Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, Canada

7.1 Introduction 7.1.1 Structures and Properties of Dendrimers 7.1.2 Multivalent Effect 7.1.3 Dendrimers as Multivalent Carbohydrate Ligands 7.2 Synthesis of Glycodendrimers 7.2.1 General Approaches to Glycodendrimer Synthesis 7.2.2 Carbohydrate-Coated Dendrimers 7.2.3 Carbohydrate-Centered Dendrimers 7.2.4 Carbohydrate-Based Dendrimers 7.3 Biological Applications of Glycodendrimers 7.3.1 Binding of Glycodendrimers to the Lectin Concanavalin A 7.3.2 Inhibition of Binding of Influenza Virus Hemagglutinin to Host Cell Sialic Acid Residues 7.3.3 Inhibition of Binding of FimH on Escherichia coli Type 1 Fimbriae to Mannose Residues on Host Cells 7.3.4 Inhibition of Cholera Toxin 7.3.5 Glycodendrimer Mimics of T-Antigen Markers on Breast Cancer 7.4 Conclusions and Perspectives References

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Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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7.1 INTRODUCTION 7.1.1 Structures and Properties of Dendrimers Highly branched structures are found widely in nature, where the branching and display of multiple terminal functionalities enable the enhancement of functions. For example, the highly branched structure of glycogen allows multiple glycogen phosphorylases to act simultaneously to cleave multiple glucose units from the polymer, thus providing a rapid source of energy for metabolic processes. In addition, a Gecko’s foot displays a highly branched network of hairs, creating a strong adhesion due to many van der Waals forces between each hair and the surface [1]. At the interface of polymer and synthetic organic chemistry, dendrimers emerged as a new class of highly branched molecules in the late 1970s and 1980s. In 1978, Buhleier and co-workers developed an iterative method for the synthesis of low-molecular-weight (MW) branched amines, which were termed cascade molecules [2]. In 1984–1985 Tomalia et al. reported conditions that were less prone to side reactions and were more suitable for repetitive growth [3]. This gave rise to the first class of molecules termed dendrimers, a word derived from the Greek word dendron, meaning “tree.” While both hyperbranched polymers and dendrimers consist of branched repeat units, they differ markedly in their preparation and resulting structures. Hyperbranched polymers are generally prepared by a noniterative polymerization procedure that results in an irregular architecture containing incompletely branched points in the structure (Fig. 7.1a) [4–6]. In contrast, dendrimers are prepared by a stepwise iterative approach that results in a regularly branched structure (Fig. 7.1b) [7, 8]. Dendrimers consist of three structural regions: (a) a core or focal point, (b) layers of branching repeat units, where each layer typically results from one stage of growth and is termed a generation, and (c) end groups on the peripheral layer.

(a)

(b)

FIGURE 7.1 Cartoons comparing the structures of (a) a hyperbranched polymer and (b) a dendrimer.

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There are several important differences between dendrimers and traditional polymers. While most syntheses of linear and hyperbranched polymers lead to a range of molecules differing in molecular weights (MWs), the iterative synthesis of dendrimers, with a focus on driving reactions to completion and even purifying intermediate molecules, leads to essentially monodisperse species. In addition, unlike linear polymers, which can theoretically be grown infinitely, the growth of dendrimers is mathematically limited owing to the exponential increase in the number of monomer units with each generation, while the volume available for these units increases with the cube of the dendrimer radius. This causes high-generation dendrimers to exhibit globular conformations and at a certain generation, sterics, known as De Gennes dense packing [9], limit regular growth and if growth is continued structural flaws will result. Finally, one of the most important differences for biomedical applications of dendrimers is that while linear polymers have only two end groups, resulting in their properties being dominated by the main-chain repeat units, dendrimers have an exponentially increasing number of end groups, resulting in the properties of dendrimers being dominated by these end groups at high generations. Most pertinent to the field of glycodendrimers is that this large number of end groups confers dendrimers with a property termed multivalency. 7.1.2 Multivalent Effect The valency of a molecule has been defined as the number of separate connections of the same kind that it can form with other molecules through ligand–receptor interactions [10]. It is proposed that multivalent interactions are prevalent in biology, such as in the adhesion of viruses and bacteria to cell surfaces and in the binding of cells to other cells. Many of these processes involve the interactions of carbohydrates with protein receptors, called lectins. While the interaction of individual carbohydrate ligands with their receptors is often weak [11], multivalency provides a means of significantly increasing the strength of the interaction. The pioneering work of Lee and Lee demonstrated that simple cluster glycosides exhibited dramatically improved binding affinities to the asialoglycoprotein receptor in comparison with the monomeric ligand [12]. Since then, there has been significant interest in the development of synthetic multivalent carbohydrate ligands in order to better understand multivalent interactions in biological systems and to potentially develop therapeutics. In general, synthetic multivalent ligands can be classified as either inhibitors or effectors. Inhibitors are those multivalent ligands that prevent receptor–ligand binding, while effectors are those that induce a cellular response upon binding. There have been several mechanisms proposed to explain the increased affinities of multivalent ligands for their receptors in the context of biological systems. For example, multivalent ligands can bind to oligomeric receptors (Fig. 7.2a). The translational entropy cost is paid with the first ligand–receptor contact and additional binding interactions can proceed with smaller entropic costs. This is commonly referred to as the chelate effect. In a variation of this scenario, multivalent ligands can also bind simultaneously to multiple receptors that are not oligomeric. This process could be facilitated by the

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FIGURE 7.2 Mechanisms by which multivalent ligands can interact with their receptors: (a) binding to multiple binding sites (chelate effect), (b) inducing the clustering of receptors, (c) occupying primary and secondary binding sites on a receptor, (d) higher local concentration of epitopes (statistical effect), and (e) steric inhibition of binding sites.

two-dimensional diffusion of receptors within a fluid membrane bilayer (Fig. 7.2b). Alternatively, some proteins have binding sites in addition to the primary binding site, which can be occupied by a multivalent ligand (Fig. 7.2c). Even when only one receptor is involved in an interaction, multivalent ligands can display higher affinities due to their higher local concentration of ligands, a phenomenon that is commonly called the proximity/statistical effect (Fig. 7.2d). This effect is caused by the slower off-rate of binding due to the close proximity of other ligands that can take the place of the first ligand after it releases. Finally, a multivalent ligand’s size and hydration shell may inhibit the interaction of other binding elements such as those on the surfaces of cells and viruses with their complementary partners (Fig. 7.2e).

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7.1.3 Dendrimers as Multivalent Carbohydrate Ligands With their highly branched structures and multiple peripheral groups emanating from a central core, dendrimers are well poised to display multivalent carbohydrates. Although a wide variety of scaffolds such as proteins, lipids, and polymers are available for the synthesis of multivalent carbohydrates, these structures pose limitations in relation to heterogeneity of their structures [13,14]. Dendrimers provide the opportunity to prepare structures as well defined as small glycoclusters, but with the sizes and valencies approaching those of glycopolymers. In addition, the generation of the dendrimer and its constituent branching units and linkers can be readily selected to tune both its valency, as well as the distance between carbohydrate ligands. At this stage, a diverse array of dendrimer backbones is accessible. For example, the poly(amido amine) (PAMAM) Starburst (Fig. 7.3a), polyester Boltorn dendrimers (Fig. 7.3b), poly(propylene imine) (PPI) (Fig. 7.3c), and polylysine (Fig. 7.3d) dendrimers are commercially available at this time, while carbosilane (Fig. 7.3e) and a wide variety of other dendrimer backbones can be readily prepared. Since their introduction by Roy et al. in 1993 [15], tremendous progress has been made in the syntheses of glycodendrimers, in understanding and optimizing their interactions with proteins, and in their development as potential therapeutics [16–28]. Key developments in glycodendrimer synthesis and several representative biological applications of glycodendrimers will be discussed in this chapter.

7.2 SYNTHESIS OF GLYCODENDRIMERS 7.2.1 General Approaches to Glycodendrimer Synthesis Like all dendrimer syntheses, the syntheses of glycodendrimers can be categorized into two general strategies: the divergent approach and the convergent approach [7, 8, 29]. In the divergent approach (Fig. 7.4a) [3, 30–33], the dendrimer is grown outward from the core by the repetition of coupling and activation steps. Reaction of functionalities on the core with a complementary group on the monomer is carried out while the peripheral functionalities on the monomer are designed to be inert to the core functionality, thus preventing uncontrolled hyperbranched polymerization. After driving this first coupling reaction to completion, the peripheral functionalities can be activated and then reacted with an additional layer of monomers. Repetition of these coupling and activation steps provides an exponential increase in the number of peripheral groups and reactions at each step. This approach is the preferred one for the large-scale industrial preparation of dendrimers because the quantity of dendrimer sample increases with each generation, and the removal of excess reagents by techniques such as precipitation, distillation, or ultrafiltration is facilitated by their differences in mass. However, the exponentially increasing number of coupling reactions required for each subsequent generation means that the number of side reactions or incomplete couplings also increases, ultimately leading to incomplete branching and flawed structures. The similarity of these flawed structures to the

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FIGURE 7.3 Commonly used dendrimer scaffolds: (a) PAMAM, (b) Boltorn, (c) PPI, (d) polylysine, and (e) carbosilane.

target molecules makes them nearly impossible to separate. Nevertheless, dendrimers prepared by the divergent approach are still quite monodisperse when compared with low polydispersity linear polymers and are suitable for most applications. The convergent approach (Fig. 7.4b), introduced by Hawker and Fr´echet, addresses many of the problems inherent to the divergent approach [34, 35]. Growth initiates from what will become the dendrimer periphery and progresses toward the core. First the peripheral groups are coupled to each branch of the monomer, while keeping the focal point of the monomer in an unreacted form. This focal point can then be activated and subsequently coupled to another monomer unit. This reaction sequence of coupling and activation continues until the desired generation is reached, and then the resulting dendritic fragments, referred to as dendrons, are finally coupled to a

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FIGURE 7.4 Comparison of the (a) divergent and (b) convergent approaches to dendrimer synthesis.

core molecule. This approach therefore only involves a small number of coupling reactions at each generation, and molecules that result from incomplete couplings can often be separated chromatographically from the desired molecules as they are sufficiently different in structure. Therefore, this approach generally affords dendrimers with higher structural homogeneity and monodispersity than the divergent approach. Nevertheless, the couplings become increasingly challenging due to steric hindrance as the dendrons approach higher generations. Furthermore, although the MW increases with each generation, the excesses of dendrons used in the couplings, incomplete couplings, and losses associated with the purification generally result in a decrease in the overall mass of material at each step, making this approach less attractive on a large or industrial scale.

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FIGURE 7.5 Three classes of glycodendrimers: (a) carbohydrate coated, (b) carbohydrate centered, and (c) carbohydrate based.

While glycodendrimers can be synthesized by divergent, convergent, or a combination of these approaches, their structures can generally be classified under three groups, according to the location of the carbohydrates in their structures. Carbohydrates can be incorporated on the dendrimer periphery (carbohydrate-coated, Fig. 7.5a), at the core (carbohydrate-centered, Fig. 7.5b), or at the branching points (carbohydrate-based, Fig. 7.5c). Their syntheses will be discussed under these headings in the context of the divergent and convergent synthetic approaches. 7.2.2 Carbohydrate-Coated Dendrimers Arguably, the most straightforward method for preparing carbohydrate-coated dendrimers involves the conjugation of carbohydrates to presynthesized or commercially available cores. The most commonly used dendrimer scaffolds for this approach include the PAMAM [36–60], Boltorn [19, 61–63], PPI [64–71], polylysine [72–81], and carbosilane [82–90] scaffolds illustrated in Figure 7.4, but several other dendrimers and dendrons such as those based on N,N-bis(3-aminopropyl)glycine (Fig. 7.6a) [91, 92] N,N-bis(3-aminopropyl)succinamic acid (Fig. 7.6b) [93], gallic acid/oligo(ethylene glycol) (Fig. 7.6c) [94–96], and 3,5-di-(2-aminoethoxy)-benzoic acid (Fig. 7.6d) [97–105] have also been divergently functionalized (Fig. 7.6). In this strategy, it is imperative that a high-yielding reaction is selected for the covalent conjugation of the carbohydrates to the dendrimer periphery to obtain homogeneous products. To this end, several key reactions have been commonly used and are illustrated in Figure 7.7. In the first glycodendrimer synthesis, reported by Roy et al., protected mannose residues derivatized with thiols were reacted with N-chloroacetylglycylglycine linkers at the periphery of a lysine dendron via an SN 2 reaction, then the sugar-protecting groups were cleaved [15]. This ligation method has also been used in several subsequent syntheses [75, 76, 92, 94, 106]. Another commonly used method involves the reaction of isothiocyanate derivatives of carbohydrates with the peripheral amine groups of various dendrimers [36–49, 54, 57, 58, 99, 107–111]. This approach has the advantage that it can be used on the

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FIGURE 7.6 Dendrimers based on the following backbone monomers have been functionalized with carbohydrates in glycodendrimer syntheses: (a) N,N-bis(3-aminopropyl)glycine, (b) N,N-bis(3-aminopropyl)succinamic acid, (c) gallic acid/oligo(ethylene glycol), and (d) 3,5-di-(2-aminoethoxy)-benzoic acid.

unprotected sugars [49, 111]. Carbohydrates have also been conjugated to dendrimers with peripheral amines via amide bond formation. For example, PPI dendrimers have been reacted with the N-hydroxysuccinimide derivatives of d-galactose and d-lactose [69,70] or directly with carboxylic acid derivatives of d-galactose [65]. Other amineterminated dendrimers have also been reacted with N-hydroxysuccinimides [73] or carboxylic acids of sugars [56, 77, 93, 97, 103–105]. An alternative method for the conjugation of sugars via amide bonds has involved the reaction of the terminal amines of PAMAM dendrimers with the lactone forms of sugars [51, 60, 112, 113]. Reductive amination has also been used to react amine-terminated dendrimers with the reducing end of sugars [66, 71, 79]. It should be noted that these last two methods result in the conjugation of ring-opened sugars. For the functionalization of

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FIGURE 7.7 Commonly used reactions for the conjugation of carbohydrates to the peripheries of dendrimers.

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carbosilane dendrimers, the most commonly used approach has been to react thiol derivatives of sugars with bromide-terminated dendrimers [82, 84, 87–90]. Over the past several years, the highly efficient Cu(I)-catalyzed click cycloaddition reaction has emerged as a very popular means of conjugating alkyne- or azide-functionalized sugars to the complementary azide- or alkyne-functionalized dendrimers. It has been used with a wide variety of dendrimer backbones including the polyesters [61], carbosilanes [85], and those based on gallic acid [95, 96] or 3,5-di-(2-aminoethoxy)benzoic acid [98, 100, 102, 114]. In addition, the chemoselectivity of this reaction allows it to be used with unprotected sugars [61, 95, 96, 100, 102]. An alternative, quite different ligation method has been the enzymatic addition of carbohydrate residues to dendrimers already bearing carbohydrate peripheries. For example, peripheral N-acetylglucosamine moieties on a polylysine dendron were converted to N-acetyllactosamines using this approach [74]. The convergent syntheses of carbohydrate-coated dendrimers differ from the divergent syntheses in that while carbohydrate conjugation is the last step of the divergent synthesis, it is the first step in the convergent approach. It involves starting with the conjugation of a simple sugar or small glycocluster to a branching unit, then finally to a core. Tris(hydroxymethyl)aminomethane (TRIS)-based glycoclusters have been a popular building block in the convergent synthesis of glycodendrimers. In the first example of this approach, the glucoside 1 was coupled to the trimesic acid derivative 2 to yield the nonavalent dendrimer 3 via a peptide coupling as shown in Scheme 7.1 [115]. It is noteworthy that it was necessary to incorporate glycine spacers between the trimesic acid core and the branching units to reduce the steric hindrance associated with the coupling. In an extension of this approach, 1 could first be coupled to the 3,3-iminodipropionic acid branching unit 4 or larger branching units, following by coupling to 2 to provide larger dendrimers such as 5 (Scheme 7.2) [115]. The same approach was applied to dendrimers with peripheral mannose residues [116]. Dendrimers with ferrocene and porphyrin cores were also prepared and their electrochemical and photophysical properties, respectively, were studied [117, 118]. A similar approach has also been carried out using unprotected TRIS-based glycoclusters, aimed at reducing the steric hindrance associated with the peracetylated sugars (Scheme 7.3) [119]. Boysen and co-workers prepared glycerol-based glycodendrimers using convergent methods. In one approach shown in Scheme 7.4, isopropylidene-protected hydroxyl ethyl mannoside 8 was reacted with methallyl dichloride (MDC) to give a mannose dimer 9 [120]. The double bond of 9 could then be ozonized and reduced with sodium borohydride, to yield the alcohol 10. After a repetition of the coupling and activation sequence, the 4-valent mannose-functionalized dendron 12 was obtained. This sequence could be further repeated to yield the 8-valent dendron. A variation of this work involved the incorporation of both mannose and galactose, where mannose was conjugated to the periphery by its anomeric center, whereas galactose was linked via its 6-position [121]. A polyphenylene-based dendrimer with peripheral glucose units was also assembled by Sakamoto and M¨ullen using a convergent approach based on Diels–Alder reactions [122]. Al-Mughaid and Grindley prepared glucose-functionalized clusters

272 SCHEME 7.1 Convergent synthesis of a nonvalent dendrimer based on a TRIS branching unit.

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SCHEME 7.2 Convergent synthesis of a dendrimer with 18 peripheral carbohydrates using both TRIS and 3,3-iminodipropionic acid branching units.

based on pentaerythritol and attached them to core molecules using Sonogashira couplings [123]. Using a metal-mediated assembly strategy, Roy and Kim conjugated GalNAc-fnctionalized dendrons to bipyridine units and then used Cu(II) to assemble two of the bipyridines [124]. Kikkeri and co-workers used a similar approach in convergently preparing dendrons with hydroxyquinoline cores and various sugars on their peripheries and used metals such as Zn(II) and Al(III) to assemble

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SCHEME 7.3 Convergent synthesis of a nonvalent dendrimer using unprotected carbohydrates.

these dendrons into fluorescent dendrimers [125]. Using a combination of divergent and convergent processes, amphiphilic glycodendrimers with GalNAc peripheries based on a p-tert-butylcalix[4]arene scaffold have also been prepared [53]. As described above, while the convergent approach is somewhat more synthetically laborious, it does present the advantage of providing structurally pure molecules. In contrast, when large preformed dendrimers are modified divergently with carbohydrates, the possibilities of uncoupled terminal groups or flaws in the backbone of the dendrimers are difficult to exclude.

7.2.3 Carbohydrate-Centered Dendrimers The natural multivalency of carbohydrates makes them suitable cores for the synthesis of dendrimers. They differ from most traditional dendrimer cores in their higher valency; however, this can be advantageous for the rapid synthesis of large dendrimers. In addition, the stereochemical properties of the dendrimer core can potentially lead to controlled orientations of the dendrimer arms in space or chiral dendrimers with chirooptical properties. Although carbohydrates have been used as pentavalent cores for the preparation of octopus glycosides [126], these molecules do not possess the backbone branching units characteristic of dendrimers. The first true carbohydrate-centered glycodendrimer, synthesized by Dubber and Lindhorst was a PAMAM dendrimer initiated from the octopus glucoside 2-aminoethyl 2,3,4,6-tetraO-(2-aminoethyl)-␣-d-glucoside (13) as shown in Scheme 7.5 [127]. The specific

275

SCHEME 7.4 Convergent synthesis of dendrimers based on glycerol.

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SCHEME 7.5 Divergent synthesis of a glucose-centered PAMAM dendrimer.

rotation values decreased with increasing dendrimer generations due to the dilution effect known for chiral dendrimers [128]. Even higher core valency was achieved using the nonreducing disaccharide trehalose as a core molecule, but structural defects caused by steric hindrance associated with the high valency arose [129]. This problem was solved by the use of longer spacers between the dendrons and the trehalose. Another interesting aspect of using carbohydrates as cores for dendrimer syntheses is that the special reactivity of the anomeric position can lead to selectively functionalized carbohydrate-centered dendrimers that would otherwise be difficult to obtain. This was demonstrated in the divergent growth of carbosilane dendrimers from a glucose core in which the aglycon moiety was not involved in the iterative dendrimer growth [130]. Convergent approaches have also been used to conjugate dendrons to carbohydrate cores. For example, trivalent mannose dendrons based on TRIS were conjugated to a penta-O-(3-aminopropyl)-d-glucoside core by thiourea linkages, providing dendrimers with 15 peripheral mannose moieties in an effort to elucidate the effects of valency, anomeric configuration of the core, and the nature of the bridging groups on their properties [131]. An isothiocyanate-armed polymannoside dendron has also been conjugated to a ␤-cyclodextrin core, and the effects on the encapsulation of molecules in the cyclodextrin cavity were studied [132].

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7.2.4 Carbohydrate-Based Dendrimers The multifunctional nature inherent to carbohydrates has made them attractive branching monomers for the synthesis of glycodendrimers. Indeed, of all the naturally occurring polymers, polysaccharides are the only ones to typically display branching. Based on the natural systems, the most fundamental design of a carbohydrate-based dendrimer involves the conjugation of each monosaccharide with two other monosaccharides through native glycosidic linkages. Colonna and co-workers have executed this approach convergently, preparing heptasaccharide 21 based on ␤(1 → 3)- and ␤(1 → 6)-linked glucopyranose units as shown in Scheme 7.6 [133]. This dendron

SCHEME 7.6 Convergent synthesis of a dendrimer based on a glucose backbone.

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was then attached to a spacer-functionalized trimesic acid core 2 to provide the dendrimer 22. The introduction of other functional groups into carbohydrate monomers provides an opportunity to form linkages other than glycosidic bonds in the dendrimer backbone. For example, Sadalapure and Lindhorst have functionalized a monosaccharide with a Boc-protected primary amine-containing aglycone and a tertiary amine having two carboxylic esters at the 6-position. Removal of the ester or Boc-protecting groups generates a reactive monomer that can be coupled in an iterative convergent manner to form dendrons as shown in Scheme 7.7 [134]. In an alternative strategy aimed at the rapid synthesis of large glycodendrimers, oligosaccharides rather than monosaccharides were used as the AB2 repeat units such that the two branching points were on different sugars [135–137]. For example, ␤-maltosyl-(1 → 6)-d-galactose was a suitable trisaccharide in which amino groups were introduced at the primary C-6 positions of the two glucose residues. As shown in Scheme 7.8, these amines were then reacted with the aldehyde group of the reducing galastose unit by a reductive amination [135]. Further iterations of this synthetic strategy were used to prepare larger second-generation dendrons and first-generation dendrimers [136] that despite their low generation were estimated to span distances on the order of 11–12 nm [135]. In an analogous approach, cellobiosylgalactose was used as the AB2 branching unit [137]. Sakamoto and M¨ullen have incorporated N-acetylglucosamine in the interior of polyphenylene dendrimers via a Schmidt glycosylation [122]. Carbohydrate dendrimers have also been synthesized by divergent approaches. For example, in a strategy where monosaccharides with spacer moieties served as the branching units, Heidecke and Lindhorst have synthesized oligomannoside mimics [138]. This synthesis was based on the activation of the allyl groups on di-O-allylated sugars by hydroboration/reduction or the radical addition of mercaptoethanol to provide alcohols, followed by the glycosylation of these alcohols with di-O-allylated mannose donors (Scheme 7.9). In an approach where multiple sugars contribute to the AB2 branching units, Ghorai and co-workers have synthesized dendrimers using glucofuranose units throughout the backbone [139]. This was accomplished by attaching three 1,2:5,6-diisopropylidene glucose molecules to a trifunctional aromatic core. Deprotection, cleavage of the 5,6-vicinal diols with NaIO4 , oxidization of the resulting aldehydes to carboxylic acids, and then coupling the carboxylic acids with a branching unit based on a trisubstituted aromatic with an amine and two protected glucofuranose units provided an iterative route for dendrimer synthesis [139]. The polyphenylene dendrimers described above were also prepared by a divergent route based on the same chemistry as was used in the convergent approach [122].

7.3 BIOLOGICAL APPLICATIONS OF GLYCODENDRIMERS As protein–carbohydrate interactions are known to mediate a wide variety of biological processes, glycodendrimers were originally developed with the aims of understanding and manipulating these processes. As such, it is natural that a wide

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SCHEME 7.7 Convergent synthesis of a carbohydrate-based dendrimer comprising noncarbohydrate-based branching units.

280 SCHEME 7.8 Convergent synthesis of a dendrimer based on oligosaccharide branching units.

281

SCHEME 7.9 Divergent synthesis of a dendrimer based on mannose and aliphatic spacers.

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variety of biological applications of glycodendrimers have been explored thus far, in an effort to elucidate structure–activity relationships and to develop new therapeutics. A thorough review of all of the possible applications of glycodendrimers is beyond the scope of this chapter. Here we will highlight several key areas where glycodendrimers have been most extensively explored thus far. The role of glycodendrimers in enhancing our understanding of protein–carbohydrate interactions using the protein Concanavalin A (Con A) as a model will be described, followed by the applications of glycodendrimers toward preventing viral and bacterial infections, neutralizing bacterial toxins, and mimicking the carbohydrate epitopes found selectively on cancer cells. 7.3.1 Binding of Glycodendrimers to the Lectin Concanavalin A Lectins, a term derived from the Latin word legere, meaning “to select,” are carbohydrate-binding proteins that are selective for their sugar ligands. While several other lectins, such as pea lectin [41, 54, 75], peanut agglutinin [51, 140], Galanthus nivalis agglutinin (GNA) [98], Vicia villosa agglutinin [53], wheat germ agglutinin (WGA) [74, 94, 110], Erythrina cristagalli lectin [74], and Limax flavus [91] have been investigated in the context of glycodendrimers, Con A has been the most thoroughly studied. It is a plant lectin, isolated from the jack bean Canavalia ensformis, and binds methyl d-mannopyranoside (Me-␣-d-Man) four times better than it binds methyl d-glucopyranoside [141, 142]. It exists as a tetramer at neutral pH, with four binding sites (one per subunit) located 6.5 nm apart (Fig. 7.8) [143]. Three different assays have been commonly used to evaluate the binding of glycodendrimers to Con A. In solid-phase binding assays, Con A labeled with an enzyme or fluorophore is added to polystyrene wells to which a mannose derivative such as yeast mannan has been attached [54, 75, 109, 131]. The concentration of dendritic ligand required to inhibit the binding of Con A to the well can be determined, providing an IC50 [50]. The hemagglutination assay involves a measurement of the minimum concentration of dendritic ligand required to inhibit the Con-A-mediated crosslinking of erythrocytes and provides relative binding affinities for a series of ligands [44]. Receptor clustering assays, including precipitation and turbidity assays, involve the formation of insoluble aggregates of Con A with the dendritic ligand, and these assays can provide data concerning the kinetics and stoichiometry of binding [50, 51, 54]. Early studies of the binding of glycodendrimers to Con A were aimed at simply demonstrating the enhanced binding of these multivalent systems relative to the monovalent carbohydrates. For example, PAMAM dendrimers functionalized with 24 d-glucose moieties were demonstrated to precipitate Con A, and a greater than 1000-fold excess of monovalent d-glucose was required to prevent this precipitation [51]. In addition, a dendrimer based on di(ethylene glycol) spacers and 3,5-dihydroxybenzoate with 6 aryl mannosides on the periphery was found to exhibit a 1.7-fold greater affinity than the optimal monovalent ligand p-nitrophenyl ␣-dmannopyranoside (pNP-␣-d-Man) [109]. The next series of studies investigated the effects of valency. For example, Pag´e and co-workers prepared polylysine dendrons

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= Carbohydrate binding site

= Protein subunit

FIGURE 7.8 Cartoon depicting the subunits of Con A and their binding sites.

having 2–16 aryl mannoside residues on their peripheries [75]. In solid-phase binding assays it was found that the dendritic molecules exhibited inhibitory potencies of up to approximately 4 relative to pNP-␣-d-Man, with a plateau of inhibition at a valency of 4. This plateau was attributed to only the mannose groups conjugated to the ε-NH2 of the lysines being accessible for binding at higher generations. In a follow-up study by the same group, a similar plateau was observed for mannose-functionalized PAMAM dendrimers at valencies of 4–8, with lower relative potencies for compounds with higher valencies [54]. Similar results were later obtained for carbohydratecentered clusters and dendrimers where a 15-valent dendrimer ligand had decreased affinity on a per mannose basis relative to 5-valent ligands, which exhibited approximately 6-fold increases in binding affinity relative to Me-␣-d-Man [131]. Therefore steric crowding at the dendrimer periphery appears to likely result in inaccessible mannose residues. Woller and Cloninger have carried out a series of studies on the binding of carbohydrate-functionalized PAMAM dendrimers to Con A. First- to sixth-generation PAMAM dendrimers were functionalized with 8 to approximately 170 mannose residues, and their binding to Con A was investigated by the hemagglutination assay. Dramatic increases in the relative binding affinity up to 660-fold per sugar were found, with increasing generations and thus valencies [44]. It is interesting to note that the lack of a plateau in the relative binding affinities seems to contradict the results described above. However, it should be noted that longer and more flexible linkages to

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mannose were used in the later systems, which may decrease the steric hindrance. In addition, larger dendrimers were also investigated. It was calculated that based on the sizes of the dendrimers and the distance of 6.5 nm between the Con A binding sites, only the third-generation and larger dendrimers were capable of spanning two binding sites on Con A and were therefore likely to bind divalently providing both chelation and statistical effects [44]. Smaller dendrimers would only be expected to exhibit statistical effects, thus limiting their relative potencies. Subsequent studies in the same group involved the synthesis of third- to sixth-generation PAMAM dendrimers with varying degrees of carbohydrate functionalization [39]. Hemagglutination assays revealed that the highest affinities for the fourth to sixth generations occurred at approximately 50% loading and decreased at higher mannose concentrations, presumably because of steric interactions. The third-generation dendrimer did not show significant increases in activity with increasing sugar functionalization because it was not large enough to span multiple binding sites of Con A. The same group also reported PAMAM dendrimers with varying ratios of mannose, glucose, and galactose on their surfaces. When the systems were optimized for high-affinity binding, the mixing of high- and low-affinity ligands led to predictable trends in lectin binding, with the possibility to attenuate the interaction in a controlled manner [37, 40]. Mannose-functionalized dendrons with valencies from 1 to 8 were attached to aluminum oxide flow-through chips and the kinetics and thermodynamics of their binding to Con A and GNA were investigated by Branderhorst and co-workers [98]. The dendrons were spaced sufficiently far apart that multiple dendrons could not bridge the binding sites on individual Con A molecules. In addition, the dendrons were not large enough to span multiple binding sites on Con A. Therefore, as expected, the kinetics of binding was similar for all dendrons and their binding constants with Con A only differed by a factor of 2, supporting monovalent binding. In contrast, the valency of the dendrons had a strong effect on their binding to GNA, a lectin comprising four subunits with three binding sites per subunit, with some of them closely spaced allowing chelation. Furthermore, Gestwicki and co-workers have used Con A as a model lectin to evaluate the effects of ligand architecture on the ligand receptor binding [50]. Several different ligand architectures were explored, including low-MW compounds, dendrimers, globular proteins, linear polymers of defined lengths, and high-MW polydisperse polymers. In a solid-phase binding assay the defined length and polydisperse polymers were found to be the most effective inhibitors. Precipitation assays revealed that the ratio of Con A molecules that were precipitated per molecule depended on the size of the ligand, with dendrimers precipitating intermediate numbers of Con A molecules per dendrimer in the range of 1:5 to 1:10 (ligand to Con A). In kinetic turbidity assays, ligands with the high valencies and epitope densities most rapidly initiated Con A clustering. Again, the dendrimers exhibited intermediate rates of Con A clustering, performing better than the globular protein, despite its similar shape. Overall, the above studies illustrate several important points concerning the use of glycodendrimers as ligands. First, the ability of the dendrimer to span multiple binding sites on a lectin has a significant effect on its binding affinity. Dendrimers that are too small to bridge multiple binding sites display only modest enhancements in binding

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affinity that can likely be attributed to statistical effects and that do not strongly depend on the dendrimer’s valency. In contrast, those capable of chelation can lead to large multivalent effects that are dependent on the valency. As a dendrimer’s size can be readily tuned by the choice of backbone and generation, it should be very feasible to manipulate the strength of binding. Second, in large dendrimers in particular, steric hindrance at the periphery of the dendrimer can inhibit the binding of carbohydrates to their receptors. Therefore, although complete functionalization is attractive from the standpoint of preparing homogeneous molecules, it does not necessarily lead to the best ligands. Finally, likely due to their intermediate sizes and valencies, dendrimers may exhibit binding affinities intermediate between those of small molecules and high-MW polymers. Despite their intermediate affinities, dendrimers are still highly attractive ligands due to the possibility of preparing reproducible and homogeneous species, an aspect that may be critical to their application as therapeutics. 7.3.2 Inhibition of Binding of Influenza Virus Hemagglutinin to Host Cell Sialic Acid Residues One of the most important examples of multivalency in biology is a microbial infection that is initiated by an adhesion. This adhesion is often mediated by multiple interactions between carbohydrates and proteins. Therefore, it has been proposed that the blocking of these interactions by carbohydrates may lead to an antiadhesion approach to therapy against microbial infections [144]. The adhesion of the influenza virus to host cells is one of the most well-studied microbial adhesion systems. It is known that all types of influenza virus contact host cells through an interaction between N-acetyl neuraminic acid (sialic acid) residues on the host cell surface and the trimeric viral lectin hemagglutinin (HA) [145], followed by endocytosis into the cell. Monovalent sialic acid can inhibit this interaction, albeit at millimolar concentrations. In order to achieve inhibition at lower concentrations, glycopolymers have been used with good results [10, 108, 146]; however, concerns over the potential toxicity and heterogeneity of polymer backbones have made dendrimers attractive targets. Indeed, the first examples of glycodendrimers, reported by Roy et al., were polylysine dendrons such as 39 (Fig. 7.9), terminated with 2–16 ␣-thiosialic acid residues [15]. On a per sugar basis, the 16-valent dendron was demonstrated to be approximately 10-fold more potent than monovalent sialic acid and thus as potent as the analogous synthetic glycopolymers in the inhibition of the hemagglutination of human erythrocytes by Influenza viruses. Several other variations on these structures have also been investigated. For example, dendrons based on N,N-bis(3-aminopropyl)glycine with 2–16 peripheral thiosialosides were prepared. Their high symmetry relative to the polylysine-based dendrons facilitated their characterization by high-field nuclear magnetic resonance (NMR) [92]. In a subsequent study, these dendrons, along with analogous dendrimers with valencies of 4–12 were investigated for their binding to Limax flavus (LFA), an animal lectin specific for sialic acid. In turbidimetric analyses, all of the dendrons and dendrimers were confirmed to crosslink LFA [91]. In a solid-phase binding assay the dendrimers exhibited an increase in inhibitory potential with increasing multivalency up to 15-fold relative to a model monovalent sugar on

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FIGURE 7.9 Chemical structure of a sialic-acid-functionalized polylysine dendron designed to inhibit the binding of influenza virus hemagglutinin to host cell sialic acid residues.

a per sialoside basis. In contrast, the tetravalent dendron was the most effective inhibitor with 32-fold greater potency than the model. Various generations of PAMAM dendrimers have been conjugated to isothiocyanate derivatives of sialic acid to provide valencies of 4–32 [58]. A steady increase in inhibitory potential resulted from increasing multivalency; however, the maximum relative potency of approximately 7-fold per sialoside in a solid phase binding assay involving LFA was lower than the tetravalent N,N-bis(3-aminopropyl)glycine dendron described above. Trivalent and nonavalent ␣-thiosialosides have also been synthesized dendrimers comprising gallic acid and oligo(ethylene glycol) [94]. For the nonamer, similar results to the above

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were found in turbidimetric analyses with LFA and WGA, but the trivalent molecules failed to form insoluble complexes with either lectin. A series of N-linked ␣-sialodendrons based on a N,N-bis(3-aminopropyl)succinic acid backbone have been synthesized on solid phase [93]. Like the S-linked sialosides, the N-linked sialosides were expected to be resistant to sialidases. This is important because in addition to HA, the other protein on the surface of the influenza virus is a sialidase that cleaves sialic acid residues from glycoproteins on the surfaces of host cells. Indeed sialidase inhibitors have been used as therapeutics in the clinical treatment of influenza [147], and the tetrameric structure of sialidase makes multivalent sialidase-resistant dendrimers possible enhanced therapeutic inhibitors [82]. A study of various sialic-acid-functionalized dendritic polymers has been carried out in an effort to determine the optimal polymer architecture for inhibiting viral adhesion [108]. Dendrimers, linear polymers, linear-dendron copolymers, combbranched, and dendrigraft polymers were investigated. In hemagglutination assays, the dendrimers were only moderately more effective than monomeric sialic acid, but the comb-branched and dendrigraft polymers were highly effective and were even able to prevent influenza infection of mammalian cells. The relatively low activity of the dendrimers was attributed to their relatively small size of 1–10 nm compared to the influenza virus (∼120 nm). Therefore, many more dendrimers per virion may be required to block virus adhesion to the target cell. This suggests that it may be worthwhile to investigate larger, higher generation dendrimers for this application. 7.3.3 Inhibition of Binding of FimH on Escherichia coli Type 1 Fimbriae to Mannose Residues on Host Cells The development of increasing numbers of bacterial strains that are resistant to antibiotics is motivating intense interest in the development of new methods for preventing and treating bacterial infections. In this context, the inhibition of bacterial adhesion to host cells is emerging as an attractive therapeutic target for the design of new antibiotics [148]. It is recognized that pathogens bearing carbohydrate recognition domains on their hairlike appendages called pili or fimbriae often bind to arrays of glycoconjugates on mammalian cell surfaces as the critical first step in host tissue colonization and biofilm formation [149,150]. FimH is the protein involved in the attachment of uropathogenic E. coli to the surfaces of bladder cells. This protein, found on the tips and along the shafts of type 1 fimbriae, contains a single binding site for mannose derivatives [151]. The E. coli bind multivalently to the bladder tissue surface via the simultaneous binding of multiple fimbriae. Through the preparation of various ␣-d-mannopyranosides, it has been established that aryl mannosides [152] such as pNP-␣-d-Man and alkyl mannosides [151] such as heptyl ␣-d-mannopyranoside are the ideal ligands with IC50 ’s in the nanomolar range in comparison with Me-␣-d-Man with an IC50 in the micromolar range. In this context, several multivalent mannosefunctionalized dendrimers have been investigated in an attempt to obtain enhanced inhibitory potencies. Early studies by Lindhorst and co-workers involved the investigation of PAMAM dendrimers such as 40 (Fig. 7.10) with mannose linked to the peripheries via

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FIGURE 7.10 Chemical structure of a mannose-functionalized PAMAM dendrimer designed to inhibit the binding of FimH on E. coli type 1 fimbriae to mannose residues on host cells.

isothiocyanates [48]. Compounds with valencies of 2–8 were investigated for their abilities to inhibit the hemagglutination of guinea pig erythrocytes. It was found that relative to Me-␣-d-Man, 2- and 3-valent molecules exhibited potencies approximately 35-fold better on a per mannose basis, but further valency increases did not improve the potencies. It was hypothesized that hydrophobic interactions involving the spacer might play a role. Later, the same group explored dendrimers based on several other scaffolds. Polyglycerol dendrimers with two or four mannose residues were evaluated against yeast mannan for binding to E. coli in a solid-phase assay [120]. These ligands performed better than Me-␣-d-Man, but none performed as well as pNP-␣-d-Man. In addition, dendrimers based on a mannose backbone with different spacer groups and bearing alkyl ␣-d-mannose functionalized peripheries with

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valencies of 2 or 4 were prepared as mimetics of cell surface oligosaccharides [138]. In a solid-phase assay based on the inhibition of E. coli binding to mannan, these ligands exhibited relative inhibitory potencies 1–2 orders of magnitude better than Me-␣-d-Man. The compounds with higher valencies and longer spacers generally performed better, a result attributed to the increased conformational flexibility and lipophilicity of the longer spacer. Lysine dendrons functionalized with 2–16 aryl ␣-mannosides were also prepared and evaluated in comparison with neoglycoproteins and 2- and 3-valent cluster ligands using hemagglutination assays and an inhibition assay involving the displacement of a radiolabeled neoglycoprotein ligand [153]. These assays revealed that the dendrimers exhibited significant affinity enhancements of up to 4-fold per mannose relative to pNP-␣-d-Man and up to 700-fold relative to Me-␣-d-Man. They increased with the valency and molecular size suggesting that they were capable of binding to multiple fimbrial sites. It was concluded from this study that the presence of multiple mannose residues that can span a distance of greater than 20 nm was important for activity. Appeldoorn and co-workers have compared small 2-valent mannose compounds with 16-valent glycodendrimers based on PAMAM and 3,5-di-(2-aminoethoxy)benzoic acid based cores as well as 21-valent glocopolymers [105]. The mannose groups were conjugated to the dendrimers by amide bonds formed between the dendrimer amines and carboxylic-acid-functionalized aliphatic spacers on the mannose. A new solid-phase assay was developed based on the inhibition of the binding of E. coli to a monolayer of T24 cells derived from human urinary bladder epithelium. All of the compounds showed enhanced affinities compared to mannose itself, with enhanced affinities generally observed for compounds with greater valencies. IC50 ’s were in the low micromolar range. The linear glycopolymers did not display any advantages over the glycodendrimers when compounds of similar sizes and valencies were compared. Overall, in the development of mannose-functionalized dendrimers for the inhibition of E. coli adhesion, molecules of relatively low valencies have generally been examined, and major multivalency effects have not been observed when the relative IC50 ’s are considered on a per mannose basis. It is proposed that the rather small enhancements are consistent with statistical effects on monovalent binding as the distances between binding sites may be too large to allow chelation [26]. To support this hypothesis, very long linear poly(p-phenylene ethynylene)s have been found to aggregate bacteria and 3500-fold higher concentrations of mannose were required to compete with this process [154]. This suggests that it will be worthwhile in the future to evaluate larger, higher valency dendrimers that have greater potential to span multiple FimH binding sites on the fimbraie. 7.3.4 Inhibition of Cholera Toxin Cholera is still a major concern in the developing world [155], and the causative agent of cholera, Vibrio cholerae, is also considered a category B terrorist threat. The cholera toxin (CT) is produced by V. cholerae and is a prominent example of a multisubunit protein that is capable of simultaneously binding to multiple carbohydrate moieties.

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FIGURE 7.11 Chemical structures of the GM1os 41 and mimics 42 and 43 for binding to cholera toxin.

It belongs to the family of AB5 toxins along with related toxins such as the heat labile enterotoxin on E. coli and the shigalike toxins. Its B subunits bind to the carbohydrate portion of the GM1 ganglioside (GM1os) 41 (Fig. 7.11) on the surfaces of intestinal cells, leading to toxin internalization and disease initiation by the enzymatically active A subunit [156]. The blocking of this initial oligosaccharide–toxin interaction can therefore serve as the basis for a new therapeutic approach. Indeed the pentameric architecture of the B subunits is ideal for the design of multivalent inhibitors as each of the five binding sites for GM1os are on the same face of the protein and are symmetrically spaced at a distance of 3.1 nm. While several multivalent inhibitors have been developed for CT and related AB5 toxins [157–160], glycodendrimers are emerging as particularly effective inhibitors. As it is challenging to prepare multigram quantities of GM1os [160], several studies have been directed toward the conjugation of simplified GM1os mimics to the peripheries of dendrons based on 3,5-di-(2-aminoethoxy)benzoic acid as the repeating unit. For example, as lactose had been shown to bind to CT at millimolar concentrations, in a mode similar to that of the terminal galactose of GM1os [161], 2- to 8-valent lactose-functionalized dendrons were prepared [101]. Binding studies based on the fluorescence quenching of a CT tryptophan residue upon ligand binding demonstrated that their potencies on a per lactose basis steadily increased with valencies up

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to 68-fold relative to monovalent lactose for the 8-valent molecule. However, their dissociation constants were in the micromolar range, higher than that of monovalent GM1os. Dendritic displays of 42, a more complex GM1os mimic, with a monovalent binding constant in the micromolar range, were also evaluated [104]. Using surface plasmon resonance (SPR) and solid-phase assays based on the inhibition of CT binding to GM1os, it was found that again the affinities increased with valencies, with the 4- and 8-valent dendrons being nearly 2 orders of magnitude more potent than the monovalent GM1os mimic. In this case, the binding constants were similar to that of GM1os. A simpler and effective GM1os mimic for multivalent displays was found to be a galactose with a long oligo(ethylene glycol) spacer (43). Ligation of these mimics to dendron peripheries of varying generations led to compounds with valencies of 2–8 [100]. Evaluation of these inhibitors using the same solid-phase assay described above revealed that their potencies increased remarkably with increasing valencies up to 2500-fold per sugar for the 8-valent dendron, reaching an IC50 similar to GM1os-C9 H18 CH CH2 . Despite the difficulties associated with its preparation, the most effective CT inhibitors have been obtained by the conjugation of multiple authentic GM1os molecules to the dendrimer periphery via elongated spacers [162]. An azide derivative of GM1os was prepared on a 100-mg scale by chemical and enzymatic synthesis steps and was conjugated to dendron peripheries by click cycloaddition reactions. It was shown using solid-phase binding assays that as the valencies increased from 2 to 8, the relative potencies per sugar increased dramatically, reaching 47,500-fold for the octavalent dendron, corresponding to a picomolar IC50 . This increase in binding affinity illustrates the strong cooperativity in the binding of these ligands to CT due to their ability to bridge multiple binding sites on the B subunits, and thus the great potential of multivalent therapeutics with this class of lectins. Overall, these results also demonstrate the importance of selecting optimal ligands from which to build multivalent ligand displays. In the future it will clearly be necessary to balance the requirements of large-scale syntheses with the binding properties of the ligands. 7.3.5 Glycodendrimer Mimics of T-Antigen Markers on Breast Cancer The aberrant expression of carbohydrates on the surfaces of cancer cells is one of the factors responsible for their metastatic behaviors and for their resistance to natural killer and cytotoxic cells. In particular, the Thomsen–Friedenreich carbohydrate antigen, ␤-Gal-(1→3)-␣-GalNAc (44, Fig. 7.12), referred to as the T-antigen (T-Ag) has been well documented as a cancer-related marker and as an important antigen for the detection, prognosis, and immunotherapy of breast carcinomas [163, 164]. For pharmaceutical applications, linear glycopolymers functionalized with T-Ag have been used in solid-phase glycosyltransferase assays for the high-throughput screening of drug candidates [165]. In addition, as receptors for T-Ag have been proposed to be present in metastatic sites such as the liver, bone marrow, and lymph nodes [166], multivalent T-Ag ligands can potentially be used to block the metastatic sites, particularly after surgery to protect against the spreading of cells that have evaded the intervention sites. Using T-Ag protein conjugate vaccines that did not contain

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FIGURE 7.12 Chemical structures of the T-antigen and a T-antigen functionalized polylysine dendron.

any of the peptide segment, mouse monoclonal antibodies JAA-F11 (IgG3) and C5 (IgM) were developed for immunohistochemical staining of breast adenocarcinomas [167], thus demonstrating that only the carbohydrate component was required for binding. The availability of these antibodies, and the pharmaceutical applications described above, have motivated the development of well-defined T-Ag-containing glycodendrimers. Toward this goal, T-Ag has been conjugated to the peripheries of several dendrimer backbones, and their binding to the above antibodies and to the known T-Ag binding plant lectin Arachis hypogaea has been evaluated. In one study, T-Ag-functionalized dendrimers with valencies of 2–6 were prepared by the conjugation of thiol and carboxylic acid derivatives to N,N -bis(acrylamido)acetic acid cores by 1,4-conjugate addition and amide bond formation, respectively [140]. Turbidimetric analyses performed with A. hypogaea confirmed the crosslinking abilities of the dendrimers. In solid-phase assays, the relative efficiencies of the dendrimers to inhibit the binding of mouse monoclonal IgG antibodies to a T-Ag-copolyacrylamide coating was determined, and it was found that the tetramers exhibited the greatest potencies, with enhancements of approximately 30-fold per T-Ag moiety. Interestingly, the hexamer showed less inhibition. In another study, a T-Ag derivative with a carboxylic acid functional handle was conjugated to the peripheries of PAMAM dendrimers to provide T-Ag glycodendrimers with valencies of 4, 8, 16, and 32 [55, 56]. In the same solid-phase assay as above, the relative efficiencies of the dendrimers increased

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with increasing valencies but were similar on a per T-Ag basis with approximately 120-fold higher affinities than the monomer. Interestingly, despite their hydrophilic character, these T-Ag-functionalized dendrimers were also found to bind effectively to hydrophobic polystyrene plate surfaces, and they could therefore be used as coatings themselves to bind to the above mouse monoclonal IgG antibody. Another series of multivalent T-Ag ligands comprised wedge-shaped polylysine dendrons with thiolated T-Ag molecules conjugated by reaction with peripheral N-chloroacetyl groups on the dendron [76]. Molecules with valencies of 2, 4, and 8 were prepared. The results of the solid-phase assay indicated that the 4-valent dendron exhibited the highest potency of approximately 75-fold per sugar, suggesting that the smaller dendrons may be better due to their minimal steric hindrance. Based on these results, a heterobifunctional dendron 45 with 4 peripheral T-Ag molecules and a biotin at the focal point was also synthesized with the aim of developing a sensor for detecting cancer cell receptors in potential metastatic sites [76]. This dendron was successfully demonstrated to bind to streptavidin-coated plates, then to subsequently capture the mouse monoclonal antibody IgG, demonstrating its bifunctional behavior. In summary it appears that T-Ag conjugates of relatively low valencies function well, at least in binding assays involving antibodies. The success of these glycodendrimers for the detection and treatment of cancer in more advanced model systems and in vivo remains to be demonstrated.

7.4 CONCLUSIONS AND PERSPECTIVES Since their inception in the mid-1990s, immense progress has been made in the synthesis and application of glycodendrimers. All possible variations on glycodendrimers including carbohydrate-coated, carbohydrate-centered, and carbohydrate-based dendrimers have been synthesized. Both divergent and convergent approaches have been well developed. Thus far, it appears that the conjugation of carbohydrates to preformed dendrimers based on noncarbohydrate backbones has been the most thoroughly explored approach. This is likely due to the feasibility of preparing them on a large scale due to the commercial availability of the dendrimer cores or their ease of synthesis. On the other hand, convergent approaches have successfully provided very monodisperse samples due to the possibility of purifying materials at intermediate synthetic steps. This may be important when highly homogeneous samples are required for structure–activity studies or certain biomedical applications. In the future, the challenge may not be the synthesis of the glycodendrimers themselves but the synthesis of certain carbohydrate epitopes for conjugation to the dendrimers, as these may be required in some instances for very high activities. In biomedical applications, glycodendrimers have provided structures and properties intermediate between those of small molecule glycoclusters and neoglycopolymers. While they can possess higher valencies similar to linear polymers, they often possess the homogeneities of small molecules. As described above, many of the dendrimers of relatively low valencies are more active than the dendrimers of higher valencies, but this depends on the application. When the binding sites are

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farther apart, or when there are many potential receptor sites, the larger, higher generation dendrimers tend to be more active. Dendrimers are also significantly different from linear polymers in their architectures. Dendrimers tend to be globular in structure, with increasing rigidity and steric hindrance at their surfaces with increasing generations. In contrast, linear polymers are often flexible, and this is relatively independent of their length. While the architectures of dendrimers more closely resemble those of proteins, interestingly they have often been found to be less active than linear polymers due to their more limited abilities to deform to span distant binding sites and to cover the surfaces of biological entities such as cells and viruses. In addition to the biological applications covered in this review, glycodendrimers have also been applied to many other medical problems such as the inhibition of the adhesion of other viruses including HIV (human immunodeficiency virus) [19, 62, 64, 65] and bacteria such as Streptococcus suis [103] and Pseudomonas aeruginosa [97]. They have also been investigated as potential vaccines [168], drug delivery vehicles [66, 72, 169], and other therapeutics. In future biological applications, it will be important to follow through with optimizing the properties of dendrimers for each given application by considering the distances between binding sites, the effects of linker length, conjugation method, and the particular carbohydrate moiety selected. For in vivo applications, it will also be critical to study in more detail the long-term fate of the glycodendrimers, such as whether they are biodegraded, excreted, or accumulated in specific tissues. It is likely that this will depend on the dendrimer backbone and on the surface residues. Overall, in a relatively short time period, carbohydrate dendrimers have made a significant impact, and time will reveal their long-term potential.

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Hultgren, S. J., Wyns, L., Klemm, P., Oscarson, S., Knight, S. D., and de Greve, H. (2005). Receptor Binding Studies Disclose a Novel Class of High-Affinity Inhibitors of the Escherichia coli fimH Adhesin. Mol. Microbiol. 55, 441–455. Firon, N., Ashkenazi, S., Mirelman, D., Ofek, I., and Sharon, N. (1987). Aromatic AlphaGlycosides of Mannose Are Powerful Inhibitors of the Adherence of Type 1 Fimbriated Escherichia coli to Yeast and Intestinal Cells. Infect. Immun. 55, 472–476. Nagahori, N., Lee, R. T., Nishimura, S.-I., Pag´e, D., Roy, R., and Lee, Y. C. (2002). Inhibition of Adhesion of Type 1 Fimbriated Escherichia coli to Highly Mannosylated Ligands. ChemBioChem. 3, 836–844. Disney, M. D., Zheng, J., Swager, T. M., and Seeberger, P. H. (2004). Detection of Bacteria with Carbohydrate-Functionalized Fluorescent Polymers. J. Am. Chem. Soc. 126, 13343–13346. World Health Organization Document (2006). Cholera 2005. Weekly Epidemiolog. Record 81, 297–308. Torgersen, M. L., Skretting, G., van Deurs, B., and Sandvig, K. (2001). Internalization of Cholera Toxin by Different Endocytic Mechanisms. J. Cell Sci. 114, 3737– 3747. Zhang, Z., Merritt, E. A., Ahn, M., Roach, C., Hou, Z., Verlinde, C. L. M. J., Hol, W. G. J., and Fan, E. (2002). Solution and Crystallographic Studies of Branched Multivalent Ligands That Inhibit the Receptor-Binding of Cholera Toxin. J. Am. Chem. Soc. 124, 12991–12998. Arosio, D., Fontanella, M., Baldini, L., Mauri, L., Bernardi, A., Casnati, A., Sansone, F., and Ungaro, R. (2005). A Synthetic Divalent Cholera Toxin Glycocalix[4]arene Ligand Having Higher Affinity Than Natural GM1 Oligosaccharide. J. Am. Chem. Soc. 127, 3660–3661. Kitov, P. I., Sadowska, J. M., Mulvey, G., Armstrong, G. D., Ling, H., Pannu, N. S., Read, R. J., and Bundle, D. R. (2000). Shiga-like Toxins Are Neutralized by Tailored Multivalent Carbohydrate Ligands. Nature 403, 669–672. Thompson, J. P., and Schengrund, C.-L. (1997). Oligosaccharide-Derivatized Dendrimers: Defined Multivalent Inhibitors of the Adherence of the Cholera Toxin B Subunit and the Heat Labile Enterotoxin of E. coli to GM1. Glycoconjug. J. 14, 837–845. Mertz, J. A., McCann, J. A., and Picking, W. D. (1996). Fluorescence Analysis of Galactose, Lactose, and Fucose Interaction with the Cholera Toxin B Subunit. Biochem. Biophysi. Res. Commun. 226, 140–144. Pukin, A. V., Branderhorst, H. M., Sisu, C., Weijers, C. A. G. M., Gilbert, M., Liskamp, R. M. J., Visser, G. M., Zuilhof, H., and Pieters, R. J. (2007). Strong Inhibition of Cholera Toxin by Multivalent GM1 Derivatives. ChemBioChem. 8, 1500–1503. Livingston, P. O., Koganty, R., Longenecker, B. M., Lloyd, K. O., and Calves, M. (1992). Studies on the Immunogenicity of Synthetic and Natural Thomsen-Friedenreich (TF) Antigens in Mice: Augmentation of the Response by quil A and SAF-m Adjuvents and Analysis of the Specificity of the Responses. Vaccine Res. 1, 99–109. Chen, Y., Jain, R. K., Chandrasekaran, E. V., and Matta, K. L. (1995). Use of Sialylated or Sulfated Derivatives and Acrylamide Copolymers of GalBeta1, 3GalNAcAlpha and GalNAcAlpha to Determine the Specificities of Blood Group T- and Tn-Specific Lectins and the Copolymers to Measure Anti-T and Anti-Tn Antibody Levels in Cancer Patients. Glycoconjug. J. 12, 55–62.

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CHAPTER 8

GLYCOSURFACES ANCA MATEESCU1,2 and MARIA VAMVAKAKI1,3 1 Institute of Electronic Structure and Laser, Foundation for Research and Technology—Hellas, Heraklion, Crete, Greece 2 Department of Chemistry, University of Crete, Heraklion, Crete, Greece 3 Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece

8.1 Introduction 8.2 Preparation of Glycosurfaces 8.2.1 Immobilization of Sugar Moieties 8.2.2 Glycopolymers: Grafting-to Approach 8.2.3 Surface-Initiated Polymerization 8.3 Characterization of Glycosurfaces 8.3.1 Spectroscopic Analysis 8.3.2 Microscopic Methods 8.3.3 Ellipsometry 8.3.4 Thermogravimetric Analysis 8.3.5 Contact Angle Measurements 8.4 Biological Applications 8.4.1 Lectin-Based Biosensors 8.4.2 Pathogen Detection 8.4.3 Specific Cell Interactions 8.4.4 Model Systems to Study Biomolecular Processes 8.4.5 Inhibitors of Viruses and Toxins 8.5 Conclusions and Future Trends References

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8.1 INTRODUCTION Carbohydrates are present on the surface of nearly every cell as free polysaccharides or as conjugates with proteins and lipids. Recently, it has been realized that their interaction with the surrounding environment is one of the most fundamental molecular recognition events [1]. Carbohydrates play a crucial role in many biochemical phenomena and are often the factors controlling the biological functions. Inflammation, fertilization, immune defense, cell growth, and cellular recognition are some of the biological functions in which carbohydrates participate through the interaction of their corresponding protein recognition receptors [2]. The sophisticated functions displayed by the natural occurring polysaccharides originate from their well-controlled structures, including molecular weight, macromolecular architecture, functional group branching, and distribution of the protein recognition groups. However, most of the naturally occurring polysaccharides have complicated structures and are challenging to synthesize and to modify. Synthetic glycosylated materials possessing structural diversity include sugarbased oligomers and polymers, dendrimers, proteins, viruses, and bilayers and have attracted great attention lately, as they exhibit similar functionalities with the natural occurring saccharides and are expected to mimic their complicated functions. These synthetic sugar-based systems can serve as simplified models of natural occurring saccharides and are of main interest with respect to very specialized applications. The participation of saccharides in biological recognition phenomena has led to the development of glycosylated drug and gene delivery carriers in which the carbohydrate receptors are used to direct the drugs to specific organ or cell targets [3–5] and of carbohydrate-sensitive biosensors to monitor saccharide molecular recognition processes [6, 7]. Due to their excellent biocompatibility glycopolymers have been proposed for use as scaffold materials in tissue engineering [8, 9], while sugarbased hydrogels, prepared by the copolymerization of a synthetic glycomonomer with a crosslinking reagent, are attractive as biocompatible water-absorbent materials [10, 11]. The polyvalency, inherent in synthetic glycosylated materials, is an important feature in their use as model systems to study and manipulate the specific molecular recognition functions of saccharides [12–14]. In the past decade it has been acknowledged that the high affinity and specificity of carbohydrate–protein interactions is achieved by multivalency, which occurs between clustered carbohydrates and protein receptors containing multiple saccharide recognition sites and has been cited as the glycoside cluster effect [15, 16]. These multivalent interactions lead to a stronger affinity and a greater specificity than the sum of successive monovalent protein–saccharide interactions. Synthetic sugar-based materials have been reported to amplify the saccharide signals similar to the natural occurring saccharides [17–20]. The ability and ease in controlling their molecular structure have made them emerge as important tools for investigating and manipulating the multivalent interaction mechanisms in biosystems. The synthesis of complex glycoconjugates or surface-immobilized carbohydrate moieties and the control of their architecture, shape, flexibility, size, valency, and

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FIGURE 8.1 Schematic illustration of glycosylated substrates.

sugar density have been under intense scientific research in the last years. Various types of substrates have been used for the multivalent display of sugars, including flat inorganic surfaces and inorganic nanoparticles, polymeric scaffolds, dendrimers, pseudopolyrotaxanes, proteins and peptides, and many more [19, 21–24] (Fig. 8.1). The grafting of carbohydrates onto a substrate is an efficient way to introduce sugar functional groups onto various supports and to obtain novel functional materials for unraveling the saccharides’ biological functions and for pursuing novel applications.

8.2 PREPARATION OF GLYCOSURFACES 8.2.1 Immobilization of Sugar Moieties There are a lot of studies in the literature on the grafting of proteins or peptides on various surfaces [25–28], whereas the grafting of carbohydrates or complex glycopolymers is rather limited. Nevertheless, the immobilization of carbohydrate molecules on flexible or solid surfaces has received a lot of attention lately, and it has become a very active field of research. Substrates varying from polymeric, proteins and peptides, dendrimers, and flat and curved inorganic supports have been used for the immobilization of sugar-based molecules, while different coupling techniques have been employed for their attachment. Moreover, the anchoring of small-molecule carbohydrates on templates offers the benefits of multivalent binding.

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Sugar-functionalized polymeric supports are immerging as promising materials for various applications. Polystyrene (PS) microgels can be conjugated with maltohexaose, a hydrophilic oligosaccharide, allowing for the site-specific hydrophilic modification of the hydrophobic polymer and the preparation of a new family of glycoconjugated macromolecular amphiphiles for use as substrates for biomedical and diagnostic purposes or as stationary phases in liquid chromatography [29]. Oligosaccharides comprising short linear starch chains were grafted onto electrostatically stabilized poly(methyl methacrylate) (PMMA) or PS latex particles, swollen with a hydrophobic monomer, by redox-initiated radical polymerization, to give sterically stabilized “hairy” particles. These nanocolloids provide interesting models for studying the role of naturally occurring polysaccharides present on the membranes of living cells [30]. Poly(ethylene terephthalate) (PET) fibers represent another attractive choice of substrate for sugar attachment. Depending on the nature of the immobilized sugar molecules, PET fibers can find potential applications in specific protein recognition and cell culture and can be used as antifouling or anticoagulant coatings. Free amino groups were introduced onto the PET fibers by treatment with appropriate diamines. Oligosaccharides such as maltose, maltotriose, and maltohexaose were next covalently grafted onto the amino-functionalized PET fibers by either reductive amination or amidation. In the former reaction, the amino groups on the surface react in the presence of a reducing agent with the aldehyde groups of the carbohydrates, while the later involves the reaction of the amino-modified fibers with sugar lactones such as maltonolactone and maltotrionolactone [31]. Immobilization techniques that preserve the activity of the biological molecules are highly desirable. The Cu(I)-catalyzed 1,3-dipolar cycloaddition of an azide with an alkyne group to form a triazole, also known as “click” chemistry, has become a very useful bioconjugation technique. Alkyne-terminated poly(ethylene glycol) (PEG)-modified inert and biocompatible glass substrates were employed as supports for the preparation of a sugar monolayer using an azide derivative of lactose as a model ligand [32]. Using the same immobilization method, glycosylated polymeric beads were prepared by the reaction of the hydroxyl groups present on the surface of the beads with an alkyne functional molecule followed by the click reaction with ␣-mannopyranoside azide to obtain sugar-functionalized colloids [33]. The presence of multiple carbohydrate moieties on appropriate scaffolds amplify the interactions of glycoside-mediated receptor targeting and are useful tools to study molecular recognition processes. Highly branched molecules, such as dendrimers, are capable of incorporating multiple sugar units and are suitable for the systematic analysis of the multivalency effect. For instance, higher generation dendrimers are large enough to span multiple binding sites of a specific protein, while lower generation dendrimers are too small for this purpose. The architectural features of dendrimers can be controlled by the choice of scaffold and the method used for its synthesis, while different carbohydrates can be immobilized onto the dendrimeric structures. Mannose-functionalized dendrimers have been prepared by coupling protected mannose units functionalized with isothiocyanate groups to an amine-terminated poly(amido amine) (PAMAM) dendrimer followed by deprotection [19]. Glucose-functionalized PAMAM dendrimers were prepared by the amide

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linkage between the peripheral amine groups and d-glucono-lactone units [34]. In a similar manner, lactose- and maltose-substituted PAMAM dendrimers were synthesized [35]. Mixtures of carbohydrates can be also immobilized onto the dendritic structure cores. To this end, mixtures of peracylated isothiocyanato-modified mannose, glucose, and galactose moieties were prepared and immobilized onto a PAMAM dendrimer, followed by deprotection [36]. These highly functionalized dendrimers were used to attenuate multivalent binding activities and are ideal models to understand more complex biological systems. Another type of dendritic structure is represented by poly(ethylene oxide) (PEO) starlike polymers based on a phosphazene core. The prepared glycoconjugates are dendrimer-like architectures obtained by the covalent interaction between acetyl-protected lactose moieties functionalized with trichloroacetimidate at the anomeric center and the PEO branches. Subsequent deprotection of the sugar hydroxy groups followed by sulfation produced a highly sulfated heparinoid mimetic glycodendrimer [37]. N,N -bis(acrylamido)acetic acid was also used as a core for the preparation of glycodendrimers. The T-antigen disaccharide cancer marker was immobilized onto the dendrimer structure by either 1,4-addition of the thiolated carbohydrate marker to the acrylamido group or by the reaction between a T-antigen acid derivative and the polyamino dendrimers [38]. Carbohydrate-displaying pseudopolyrotaxanes threaded onto linear polymer chains represent another class of multivalent neoglycoconjugates. Cyclodextrins (CDs), a series of oligosaccharides with a hydrophobic and hollow truncated cavity, are employed to generate polypseudorotaxanes through complexation with synthetic polymers forming supramolecular structures with a distinctively dynamic presentation of the carbohydrate units. The CDs were synthesized by a coupling reaction between a lactosyl propionic acid derivative and a monoamino cyclodextrin [39]. The lactose containing ␣- and ␤-CDs have been threaded onto poly(propylene glycol) or polytetrahydropyran chains in which the individual CDs slide along and rotate around the polymer axes to alter the orientation of the sugar moieties. In a similar manner, a lactoside-displaying CD was threaded onto linear polyviologen chains [40]. A more complex architecture, which represents an interesting alternative for studying the recognition properties of natural saccharides, consists of CD–glycodendrimer conjugates. Dendritic ␤-cyclodextrin derivatives bearing multivalent mannosyl groups and displaying a variety of carbohydrate valency were synthesized by the reaction between isothiocyanate and amine-functionalized building blocks. The obtained architecture has great potential for use as a sugar-directed delivery system of drugs to specific saccharide receptors present on biological surfaces [20]. Polypeptides and polynucleotides are considered to be excellent scaffolds for sugar residues due to their rigid and regular chain structure while they allow the presence of multiple carbohydrate recognition elements and provide a specific distance between the sugar units. The polymerization of glycosylated tripeptides in which the sugar group was linked covalently to the peptide backbone, allowed the construction of a three-dimensional arrangement of d-glucosamine residues along the ␣-helical protein backbone [41, 42]. Moreover, aminooxylated carbohydrates were successfully grafted onto peptide scaffolds bearing aldehyde groups and led to the preparation of carbohydrate oligopeptide conjugates [43]. Deoxyribonucleic acid (DNA), a

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conformationally rigid scaffold, has been also used for the attachment of lactose and cellobiose molecules, however, the immobilization of carbohydrates onto DNA without affecting its structure and stability is not trivial. One way to achieve this is the use of the diazo-coupling method upon which DNA is selectively modified with biotin derivatives at the 8-position of the guanine base. Benzenediazonium derivatives bearing lactose and cellobiose groups were subjected to diazo coupling to naturally occurring DNAs without affecting the higher-order structure of the DNA and led to DNA–carbohydrate conjugates that mimic glycosylated DNA in nature [44]. The functionalization of curved inorganic surfaces with small carbohydrate molecules or oligosaccharides has attracted a growing interest lately. Gold nanoparticles became the focus of attention in the biomedical field, with most applications relying on the use of hydrophilic PEG-stabilized gold nanoparticles [45]. The functionalization of gold nanoparticles with aldehyde-modified PEG-thiol chains provided the possibility for further covalent attachment of small sugar moieties such as lactose and their use in the immobilization of specific proteins [17]. Besides the covalent attachment of sugar-based molecules onto solid substrates, their noncovalent immobilization has been also extensively reported. However, one must keep in mind that the noncovalent attachment of molecules often results in low surface coverage and to chain desorption from the surface. Biomembrane mimetic copolymers based on phosphorylcholine-bearing galactose side chains were prepared by radical polymerization and were coated on PET substrates by solvent evaporation. The synthetic membranes were found to behave in a similar manner to the outer surface of natural membranes [8]. The self-assembly of glucose- or galactose-containing glycopolymers on hydrophobic micropatterned surfaces created on silicon substrates by photolithography was driven by the interaction of the hydrophobic polymer backbone with the substrate and led to the development of carbohydrate microarrays for cell cultivation and protein analysis [12]. An attractive scaffold for the immobilization of glycopolymers are carbon nanotubes (CNTs). The use of CNTs in biological systems is limited due to their cytotoxicity, thus new strategies to reduce their toxicity are required. An elegant method to render the CNTs nontoxic is the grafting of sugar-containing polymers on their surface. A biomimetic polymer containing acetylgalactosamine residues, designed to mimic cell surface mucin glycoprotein has shown great potential in reducing the toxicity of the CNTs [46]. A lipid was introduced at one end of the mucin mimic polymer to enable the surface modification of the CNTs by self-assembly via hydrophobic interactions [47]. However, besides the hydrophobic interactions, electrostatic forces have been often used for the surface immobilization of glycopolymers by the so-called layer-by-layer (LbL) technique [48]. This technique is based on the self-assembly of oppositely charged polyelectrolytes and results in the preparation of thin multilayer films on both planar (quartz slides, PET films, silicon wafers) and curved (inorganic and polymer colloids) surfaces. A cationic galactose-bearing branched copolymer was alternated with anionic polymer chains onto PS particles [49]. Following, the removal of the colloidal templates bioactive microcapsules were formed that are of great interest for the encapsulation and release of molecules of biological importance.

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8.2.2 Glycopolymers: Grafting-to Approach The anchoring of small sugar moieties on various scaffolds opened the path for the immobilization of more complex carbohydrate-based materials. Sugar-containing polymers, known as glycopolymers, are of particular interest due to the presence of a high density of functional groups and can be considered as useful tools for understanding the recognition abilities of natural saccharides. Glycopolymer chains anchored on a surface, in close proximity to one another, are forced to adopt a stretched conformation and to avoid the formation of entanglements, which are reported to lower the protein recognition properties of glycopolymers in solution [50]. Therefore, endanchored surface-immobilized glycopolymers are very attractive model systems to study the carbohydrate–protein interactions. The grafting-to method is frequently used to attach glycopolymer chains by one end onto various scaffolds. In this approach, end-functionalized glycopolymer chains react with the surface functionalities of a suitable substrate under appropriate conditions (Fig. 8.2). The method is limited by the crowding of the chains at the surface, which hinders the diffusion of more chains and lowers the density of the anchored sugar moieties. Nevertheless, due to its rather simplistic experimental design, the grafting-to technique proves to be a suitable method for the grafting of glycopolymer chains on various substrates. The devolvement of protein–glycopolymer conjugates by the grafting-to method has received a lot of attention lately because the protein component provides welldefined ligand binding sites and enzymatic activity, while the glycopolymer can specifically bind to proteins and cells. These types of conjugates have great potential as therapeutic agents or biological probes. Model thiol-containing protein bovine serum albumin (BSA) has served as a substrate for site-specific coupling with well-defined maleimide-terminated neoglycopolymers bearing mannose moieties [23]. These mannoside BSA structures are proposed for use to trigger pathogen cell detection. On the other hand, viruses are intriguing scaffolds for the attachment

FIGURE 8.2 Schematic representation of the grafting-to technique for the preparation of end-grafted glycopolymer chains onto various substrates.

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of sugar molecules due to the presence of multiple binding sites that lead to the formation of glycodendrimer-like structures. Virus particles modified with azide groups were condensed with an alkyne-containing glucose-based polymer prepared by a controlled/“living” radical polymerization, namely atom transfer radical polymerization (ATRP) to produce glycopolymer–virus conjugates displaying polyvalent binding properties [51]. These novel species are very attractive for both diagnostic and therapeutic applications. Inorganic silica particles have been employed as a substrate for the immobilization of glycopolymers with potential use in the separation and analysis of biological active molecules (i.e., proteins). Well-defined end-functionalized glycopolymers of low polydispersity bearing acetyl-protected glycomonomer repeat units were prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. The obtained dithioester end-functionalized polymer was grafted onto ␥ -methacryloxypropyl-trimethoxy modified silica particles followed by deprotection in a second step to reveal the pendant lactose residues [24]. The versatility of the grafting process and the well-defined polymer structure obtained by the controlled polymerization technique provide an effective route for the synthesis of advanced functional hybrid materials. The avidin (streptavidin)/biotin-based conjugation can be also used to introduce biotinylated sugar-based polymers on avidin/streptavidin-functionalized substrates. Due to the mild reaction conditions and simple washing and purification steps, the potential damage to the surface-bound ligand units upon the conjugation process is highly reduced. Streptavidin-derivatized patterned PET membranes incubated in a solution of biotin-terminated glycopolymers containing lactose moieties prepared by cyanoxyl-mediated free radical polymerization represent a reliable method to prepare glycosurface arrays of varying carbohydrate type and density [52]. Similar polymer chains can be immobilized onto streptavidin-functionalized polymeric lipid films to obtain uniform carbohydrate coatings that mimic the surface of cells [53]. 8.2.3 Surface-Initiated Polymerization The chain length and the molecular structure of sugar-based conjugates as well as the distribution of the carbohydrate recognition groups can greatly influence their recognition activity in solution. On the other hand, the surface coverage of active carbohydrates attached on supports has been shown to play an important role in protein binding [54–57]. As a consequence, the synthesis of glycosylated surfaces with well-defined macromolecular architectures and control over the surface grafting densities is highly desired. As mentioned above, the grafting-to technique is experimentally simple but suffers from intrinsic limitations such as lack of control over the chain structure and grafting density. In the recent years, the grafting-from method in tandem with controlled/living polymerization techniques have become a viable alternative to synthesize polymer chains with controllable lengths and appropriate grafting densities, while possessing a variety of functionalities. The graftingfrom technique involves the immobilization of initiator molecules onto a scaffold followed by in situ surface-initiated polymerization of an appropriate monomer

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FIGURE 8.3 Schematic representation of the grafting-from approach for the preparation of glycopolymer brushes.

(Fig. 8.3). This approach allows the synthesis of uniform polymer layers of high, grafting density, with tuneable thicknesses via molecular weight control [58]. If the grafting density of the polymer chains is sufficiently high, they become crowded and stretch away from the surface to avoid overlapping, and these polymer chains are termed polymer brushes. The preparation of polymer brushes via surface-initiated polymerization requires a uniform monolayer of initiator molecules on the target surface. The Langmuir–Blodgett, the LbL, and the self-assembled monolayers (SAMs) are among the most widely used methods for the immobilization of initiator molecules onto different substrates. SAMs are particularly attractive because they allow the preparation of high-density initiator layers with a well-defined initiation mechanism [59]. Controlled/living radical polymerization techniques such as ATRP and RAFT have been successfully applied to prepare well-defined surface-attached polymer chains of different functionalities. The basic mechanism, which is common in these polymerization techniques, is the equilibrium between the growing free radicals and the dormant species and affects the molecular weight and the molecular weight distribution of the resulting polymer. Both polymerization techniques rely on the use of appropriate vinyl glycomonomers for the preparation of sugar-based polymer brushes with control over the polymer molecular weight, the molecular weight distribution, and the grafting density. The surface-initiated ATRP of sugar monomers was investigated on a variety of substrates, and the obtained glycopolymer brushes are interesting model systems to study and understand the carbohydrate–protein interactions due to their well-defined macromolecular characteristics. Two different approaches have been employed for the preparation of glycopolymer brushes. The first method involves the polymerization of a protected glycomonomer followed in a second step by deprotection to obtain the sugar moieties. Using this strategy, well-defined glycopolymer brushes of high grafting densities were prepared on silicon substrates modified with

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a silane-functionalized ATRP initiator, using the protected methacrylate monomer [3-O-methacryloyl-1,2:5,6-di-O-isopropylidene-d-glucofuranose (MAIpGlc)]. Next deprotection of the isopropylidenyl groups by formic acid afforded the glucose-based grafted polymer chains [21]. Similarly, the synthesis of glycocylindrical polymer brushes and linear glycopolymer chains from functionalized CNTs using this protected glycomonomer was reported [60, 61]. Finally, inorganic nanoparticles are employed for the preparation of glycosylated organic/inorganic hybrids. Silsesquioxane nanoparticles modified with ATRP initiator molecules were used as macroinitiators for the polymerization of the protected MAIpGlc glycomonomer. Following the deprotection reaction, well-defined glucosebased polymer–inorganic hybrid stars were formed that are attractive for potential applications in optics and bioscience [62]. The second approach involves the direct synthesis of glycopolymer brushes without the requirement of using the protection–deprotection strategy. Glycopolymer chains bearing glucose moieties were synthesized from polypropylene microporous membranes by a combination of ultraviolet (UV)-induced graft polymerization to functionalize the polymeric substrate and ATRP to polymerize the glycomonomer. Water has been shown to have a significant acceleration effect on the polymerization rate, however, it induces a loss of control over the polymerization. The addition of copper(II) deactivator in the polymerization mixture has been also shown to exert some control on the polymer growth [63]. Well-defined sugar methacrylatebased homopolymer brushes based on d-gluconamidoethyl methacrylate (GAMA) and 2-lactobionamidoethyl methacrylate (LAMA) were prepared from functionalized gold substrates. A good control of the polymerization was achieved in mixed methanol–water solvent media in the presence of deactivator species [64]. An increase in the dry film thickness of the grafted chains was found as the methanol content of the reaction mixture increased, which suggests the suppression of the termination reactions as the polarity of the solvent decreases. Initiator-modified titanium substrates were used for the preparation of dense polyGAMA brushes. The glycopolymer brush thickness increased almost linearly with the polymerization time for a short induction period after which the brush thickness reached a plateau. This was attributed to termination reactions that occur due to the high concentration of reactive chain ends or to the burial of the chains in the growing polymer brush [65]. Metallic curved surfaces, that is, gold nanoparticles, can also serve as supports for polysaccharide attachment by the grating-from technique. The immobilization of a disulfide ATRP initiator followed by the surface-initiated polymerization of LAMA has led to the development of galactose-functionalized gold particles that show great potential in biosensing applications [66]. Besides ATRP, RAFT polymerization has also emerged as a promising technique for the controlled growth of sugar-based polymer brushes. Grafted glycopolymers of narrow polydispersity comprising of N-acryloyl glucosamine monomer repeat units were prepared on silicone substrates [67]. The RAFT agent was immobilized on an amine-functionalized surface via covalent attachment using the Z-group approach, followed by the direct polymerization of the deprotected glycomonomer. The glycopolymer brushes were successfully used for chain extension with a

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temperature-responsive monomer, thus confirming the living character of the polymerization and resulting in the formation of thermoresponsive glycopolymer brushes. The development of controlled/living radical polymerization techniques has opened the path for the synthesis of a new class of glycopolymer architecture, that of branched polymeric materials that exhibit multiple sugar moieties. Hyperbranched glycopolymers were synthesized from functionalized CNTs and silicon wafers by surface-initiated self-condensing atom transfer radical copolymerization of a methacrylic inimer and a protected glucose-based acrylate monomer, followed by deprotection [61, 68]. An interesting scaffold that presents a multivalent display of biological ligands are the pseudopolyrotaxanes. Pseudopolyrotaxanes were used as ATRP macroinitiators for the polymerization of GAMA, leading to the preparation of supramolecular and biomimetic polypseudorotaxane/glycopolymer biohybrids [69]. These triblock copolymers form hydrophilic nanoparticles in water that are potentially very useful for fabricating targeted drug delivery systems owing to the specific sugar–protein recognition and their enhanced permeability and retention in living systems. Porous structures such as polypropylene microporous membrane (PPMMs) posses a relatively large surface area for tethering glycopolymer chains, and, therefore, high sugar densities can be achieved. Microporous membranes bearing sugar moieties have been proposed for use as chromatographic supports for the isolation of proteins with specificity for different sugar moieties and as lectin and enzyme immobilization supports. UV-induced graft polymerization was used to introduce sugar-containing polymers on polypropylene membranes using ␣-d-allyl glucoside as the glycomonomer. This method allowed the preparation of glycosylated surfaces of relatively high valency and some control over the sugar density by adjusting the glycomonomer concentration, the irradiation time, and the photoinitiator concentration [50]. Alternatively, the grafted glycopolymer layer can be formed by UV-induced graft polymerization of acrylamide monomer followed by the transformation of the amide units to primary amine groups and the subsequent coupling of carbohydrate lactones to the primary amino groups [70]. A direct method to introduce amine groups on the surface of the membranes is by UV-induced graft polymerization of 2-aminoethyl methacrylate hydrochloride. The glycopolymer layer was obtained by appropriate coupling of sugar molecules in the lactone form with the amino groups of the polypropylene surface [22]. Finally, growing alkyne functional polymer chains from initiating sites immobilized on the polymer beads afforded clickable PS core–polyalkyne shell particles. By the click reaction with ␣-mannopyranoside azide, mannose-functionalized colloids were prepared. The developed strategy is applicable for various sugar moieties and can produce materials with applications in different fields, including chemical sensing, responsive surfaces, and affinity chromatography [33].

8.3 CHARACTERIZATION OF GLYCOSURFACES The analysis of thin layers grafted on various substrates is challenging due to their reduced dimensions. Nevertheless, a number of spectroscopic, microscopic, and

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optical analytical techniques have been successfully employed to study and characterize thin polymer films. This section focuses on the importance of the combination of a variety of analytical methods to fully characterize the glycosylated surfaces. The applicability of each technique depends on the composition, the structure, and the geometry of the surface to be analyzed. The continuous development of more accurate surface-sensitive methods will certainly aid the characterization and the analysis of the glycosurfaces in the near future. 8.3.1 Spectroscopic Analysis Spectroscopic techniques such as Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopies (XPS) are reliable tools for determining the chemical composition of thin layers, even at monolayer thickness, and have been used extensively for the characterization of glycosurfaces and glycoconjugates. FTIR is often employed to investigate changes in the surface chemical structure, before and after its modification with the carbohydrate. The coupling of d-gluconolactone monomer on microporous membranes functionalized with amino groups has been confirmed by FTIR spectroscopy. Following the coupling with the sugar moieties, a new peak at 1510 cm−1 attributed to the amide link formation and the appearance of a broad absorption peak around 3300 cm−1 assigned to the OH stretching vibration of the sugar hydroxyl groups proved the successful coupling [70]. The grafting of polyGAMA brushes on microporous membranes was confirmed by the appearance of additional peaks in the IR spectrum ascribed to the amide I and amide II bands and to the stretching vibration of the N H bond of the GAMA monomer and the broad absorption band assigned to the OH stretching vibration [63]. Similarly, the successful growth of polyGAMA and polyLAMA polymer brushes on initiatormodified gold substrates was confirmed by the appearance of the sugar peaks in the spectrum assigned to the amide I, amide II, and OH vibrations of the sugar monomer repeat units. Furthermore, the OH stretching vibration of the polyLAMA brushes was significantly more pronounced than that observed in the spectrum of the polyGAMA-grafted chains and was attributed to the presence of a larger number of hydroxyl groups on the polyLAMA monomer repeat units [64]. In some cases FTIR spectroscopy has been used to confirm the successful deprotection of grafted sugar-based polymer chains to give the desired sugar moieties. The cleavage of the isopropylidenyl groups to obtain the hydroxyl groups of the carbohydrate moieties was monitored. The successful deprotection was confirmed by the disappearance of the absorption bands of the C H stretching and deformation modes, the C O stretching of the isopropylidenyl group, and the appearance of the wide absorption band attributed to the OH stretching vibration [21, 68]. X-ray photoelectron spectroscopes is a very powerful technique that can give valuable information on the relative abundance of atomic species and the presence of atoms in different oxidized states. The appearance of peak components at certain binding energies in the XPS spectrum provides insight in the successful attachment of molecules on surfaces. The adsorption of a lactose-substituted styrene homopolymer on patterned silicon substrates was studied by XPS. The appearance of two extra

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peaks in the spectrum, assigned to the carbon atoms of the C O and O C O groups, after the adsorption of the glycopolymer, indicated its successful grafting on the surface [9]. The surface-initiated polymerization of amide-based glycomonomers on microporous membranes was verified by the appearance of a N1s peak in the XPS spectrum attributed to the amide groups and the increase of the intensity of the oxygen peak due to the hydroxyl groups of the glycopolymer [63]. Similarly, the attachment of glycopolymer chains onto silica particles was verified by the appearance of three peak components in the C1s peak attributed to the carbon atoms of the O C O, C O, and CH2 groups of the grafted polymer [24]. The deprotection of the sugar units can be also monitored by XPS by comparing the chemical composition of the grafted chains before and after deprotection. The hydrolysis of the isopropylidene groups of branched and linear glycopolymer brushes was confirmed by the increase of the oxygen content compared to the carbon peak attributed to the hydroxyl groups of the deprotected polymer brushes [68]. The agreement between the experimentally determined carbon-to-oxygen ratio and the theoretically calculated value can be used to assess the degree of deprotection. On the other hand, nuclear magnetic resonance (NMR) spectroscopy provides important information about the molecular structure and has been successfully employed to probe the chemical structure and verify the successful synthesis of various glycoconjugates. The target molecular structure of dendritic cyclodextrins bearing multivalent mannosyl ligands and glycodendrimers based on an iminobis(propylamine) core was verified by NMR spectroscopy [20, 71]. The technique can be also used to determine the degree of carbohydrate loading on a glycoconjugate. The high degree of carbohydrate functionalization was confirmed from the 1 H-NMR spectra of lactoseand mannose-functionalized dendrimer-like PEO and PAMAM dendrimers, respectively [19, 37]. The deprotection of cylindrical polymer brushes possessing protected sugar methacrylate side chains was studied by NMR. After the deprotection, the peak attributed to the isopropylidene protons of the protected groups disappeared and a broad signal attributed to the anomeric hydroxyl groups of the sugar moieties appeared, raveling the successful synthesis of the glycopolymer brushes [60]. Mass spectroscopy is an attractive analytical tool employed in the measurement of the molecular mass of glycoconjugates. The molecular weight of glycodendrimers was determined by the matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF) technique and allowed the calculation of the number of sugar moieties bound on the surface of the dendrimers, which is very important because it provides useful insights regarding the multivalent binding of proteins. An accurate quantitative determination of the relative amounts of mannose and hydroxyl groups immobilized on PAMAM dendrimers proved the preparation of a series of mannose/hydroxyl-functionalized dendrimers with a varying number of sugar moieties [56]. Moreover, the mannose loading of PAMAM dendrimers was found to decrease from 100% for the first two generations to 67% for the sixth-generation dendrimers [19]. The amount of grafted polymer on a surface can be determined quantitatively by colorimetric titrations. Carbohydrate-modified PET fibers were analyzed by a sugar-specific colorimetric method based on the spectroscopic determination of the

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colored product formed by the reaction of the hydrolyzed sugar moieties with a phenol/sulfuric acid mixture [72]. Other spectroscopic techniques including UV–Vis spectroscopy and circular dichroism (CD) has been used in the characterization of the glycoconjugates. UV–Vis spectroscopy confirmed the successful coupling of carbohydrates on DNA strands by the appearance of an absorption band, attributed to the azophenyl group of the modified sugars, in the spectrum of the DNA–carbohydrate conjugate [44]. CD spectroscopy can be very useful to help elucidate the structure of carbohydrate–biomolecule conjugates. DNA–carbohydrate conjugates with a carbohydrate content lower than 25% exhibited a B-type conformation similar to that of the native DNA, indicating that the modification reaction did not affect the DNA conformation. However, a higher carbohydrate substitution induced a change in the DNA conformation to an A-type duplex [44]. 8.3.2 Microscopic Methods Microscopic techniques such as atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) can monitor the formation of thin polymer layers on a variety of scaffolds. AFM or scanning force microscopy (SFM) can probe the surface morphology, the roughness, or the phase segregation on surfaces. AFM experiments performed on native PET fibers before and after carbohydrate immobilization provided useful information about the surface morphology. The native fibers were homogeneous, whereas a nanostructured surface was observed following the attachment of the sugar molecules [72]. AFM is also a powerful tool to characterize linear, hyperbranched, or cylindrical polymer brushes. The surface roughness of glycopolymer brushes grafted on modified silicon and gold substrates was found below 1 nm by AFM, suggesting the preparation of very smooth, homogeneous glycopolymer layers [21, 64]. Glycopolymer chains grafted from CNTs were identified by comparing the AFM images of the initiator-modified CNTs to those of the glycopolymer-grafted CNTs. A cylindrical nanowire-like morphology was found for the initiator-modified CNTs, while a fuzzy structure around the tube was observed for the polymer-grafted CNTs. Moreover, the width of the tube was larger than its heigh, indicating the successful grafting of the glycopolymer chains on the surface of the CNTs [61]. Glycocylindrical brushes showed characteristic wormlike structures with a very high aspect ratio of the length of the backbone to that of the side chains [60]. Grafted hyperbranched glycopolymers from silicon substrates have shown irregular protrusions distributed throughout the copolymer surface with the surface roughness decreasing upon increasing the comonomer ratio. This was attributed to the decrease of the degree of branching with the increase in the comonomer ratio, which leads to smoother surfaces [68]. Glycopolymerinorganic hybrid stars can be also characterized by AFM. The appearance of spherical isolated particles indicates the formation of uniform and well-defined hybrid stars [62]. Finally, AFM allowed to study the aggregation behavior of glycosylated peptides immobilized on surfaces from different solutions. Globular particles with a relatively narrow size distribution suggest a high degree of dispersion, while larger particles indicate a high degree of aggregation in solution [42].

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Scanning electron microscopy gives insight into the three-dimensional and the surface morphology of materials and has been employed in the characterization of many glycosylated conjugates and surfaces despite its deficiency to probe nonconductive polymer samples. Native PET fibers exhibited a rather homogeneous morphology compared to their carbohydrate-grafted analogs, which showed irregular structures on the surface, attributed to the grafted sugar molecules [72]. On the other hand, nacent PPMMs showed a relatively high porosity with a small pore size by SEM, while as the surface of the membrane was covered gradually with a glycopolymer film, the surface pores became blocked and the porosity decreased evidently with the increase of the polymer film density [50]. Finally, a continuous polymer phase covering the nanowire-like morphology of the pristine CNTs was attributed to the grafting of the glycopolymer brushes onto the CNTs [61]. SEM can be also used to determine the size of the glycopolymer layer onto nanoparticulate surfaces. The increase in the diameter of gold particles following their functionalization with glycopolymer chains gives a good measure of the glycolayer thickness [66]. Similarly, a good agreement of the average diameter of glycopolymer/silsesquioxane hybrid stars calculated by SEM to the size determined by gel permeation chromatography/multi-angle light scattering (GPC/MALS) and dynamic light scattering (DLS) measurements was found, verifying the efficacy of the technique [62]. Transmission electron microscopy is another microscopic technique that can be used to image grafted polymer layers provided there is enough contrast between the scaffold and the polymeric component. Carbohydrate-grafted CNTs and silica nano-objects provide such a contrast and are thus adequately characterized by TEM. Linear and hyperbranched glycopolymers grown from CNT surfaces showed a polymer shell around the CNTs following the attachment of the linear or hyperbranched brushes, which was absent in the initiator-immobilized CNTs [61]. Cryo-TEM measurements are particularly useful as they allow the detection of the original shape and size of the polymer in solution, by the vitrification of the sample before the measurement. The cryo-TEM images of deprotected glycocylindrical brushes showed a wormlike cylinder shape, [60] while a spherical shape was observed for sugar-based polymethacrylates grafted from silsesquioxane nanoparticles [62]. 8.3.3 Ellipsometry Ellipsometry is extensively used to determine the dry or wet film thickness of the adsorbed or grafted polymer layer. Glycopolymer films adsorbed on patterned silicon ˚ were measured by ellipsomsurfaces with dry thicknesses between 15 and 20 A etry [12], while lower thicknesses, between 3 and 9 nm, were found for polymer brushes comprising protected sugar methacrylate monomer repeat units on silicon substrates [21]. A maximum dry film thickness of 63 and 40 nm was determined by ellipsometry for polyGAMA and polyLAMA brushes, respectively, synthesized on initiator-modified gold substrates [64]. Similarly, polyGAMA brushes with a maximum film thickness of 30 nm has been reported on titanium substrates [65]. Besides, ellipsometry can be also used to monitor the successful deprotection of glycopolymer brushes. The decrease in the thickness of the grafted layer following deprotection for

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both linear and branched glycopolymer brushes was attributed to the removal of the bulky protecting groups leading to the formation of the desired sugar-based brushes [68]. Finally, the swelling of the glycopolymer brushes is determined by liquid-phase ellipsometry. An increase in the film thickness from 30 to 85 nm for the polyGAMA brushes prepared on titanium substrates was observed when ellipsometry was performed in a water-swollen film suggesting the hydration of the brush, which causes the stretching of the polymer chains away from the surface [65]. 8.3.4 Thermogravimetric Analysis Thermogravimetric analysis (TGA) has been successfully used to monitor the changes in weight of the conjugates before and after the grafting of the sugar moieties. The glycopolymer content of glycosylated CNTs was found to increase with the polymerization time, suggesting the control of the grafting process [61]. Similarly, the amount of glycopolymers grafted onto silane-modified silica gel particles was determined by TGA. An increase in the weight loss of the glycopolymer-grafted silica particles compared to that of the silane-modified particles was found, indicating the successful attachment of the sugar-based polymer [24]. 8.3.5 Contact Angle Measurements Contact angle measurements are a simple and convenient way to determine changes in the wetting characteristics of surfaces. The immobilization of carbohydrates on surfaces leads to the formation of a hydrophilic layer due to the hydroxyl groups present on the sugar moieties. This results in a decrease of the static contact angle of water droplets on the surface as was verified for glycopolymer brushes grafted on PPMMs. The change in the surface hydrophilicity can be controlled by the degree of glycopolymer grafting and has a significant effect on the separation properties of the membranes [73]. A similar increase in hydrophilicity has been reported for sugar-modified gold and titanium surfaces, which was interestingly found to depend on the hydrophilic nature of the monomer used in the polymerization [64, 65]. Such glycosurfaces are very attractive for use as biocompatible, hydrophilic surfaces in tissue engineering and the development of implants.

8.4 BIOLOGICAL APPLICATIONS The interactions of surface-immobilized carbohydrates with biologically active systems such as lectins, viruses, bacteria, or cells can yield useful information about the naturally occurring saccharides and display opportunities for their use in various applications. Due to the diversity of the potential applications in which glycosurfaces are employed including biosensors for protein and pathogen detection, scaffolds for tissue engineering, multivalent inhibitors of viruses and toxins, models for natural occurring biological systems, stationary phases for chromatography, and carriers for drug delivery, research in this area has increased dramatically over the past few years.

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8.4.1 Lectin-Based Biosensors One of the most attractive applications of synthetic glycoconjugates is their use as monitoring systems for studying the carbohydrate recognition processes. The carbohydrate–protein recognition processes occur through specific interactions between the saccharides found on the cell surface and proteins with carbohydratebinding domains known as lectins. Lectins constitute a broad family of proteins, frequently found on the surface of the cells, which are involved in various biological processes ranging from the regulation of cell adhesion to glycoprotein synthesis. They have the ability to specifically and noncovalently bind to carbohydrates, and this characteristic has been exploited as the basis for biosensor design [74]. Most lectins are oligomeric molecules with several sugar-binding sites and are able to decode the carbohydrate-encoded information and distinguish among a variety of sugar molecules. Various detection methods based on lectin–carbohydrate recognition have been developed. For instance, cyclic voltammetry can be used to detect the carbohydrate molecular recognition processes by immobilizing galactosamine-based SAMs on a gold electrode and monitoring their interaction with the soybean agglutinin lectin using ferrocyanide ion as a marker [6]. Changes in the fluorescence signal are often employed to detect such interactions with appropriate carbohydrate or lectin labeling. The binding of the lectin Concanavalin A (Con A) onto ruthenium-based glycoclusters possessing mannose units was detected by monitoring the changes in the luminescence intensity [7]. The fluorescence intensity from a mobile fluorophore-labeled Con A, which can bind competitively and reversibly to immobile pendant glucose moieties within colored Sephadex beads, was used to give insight into the saccharidebinding phenomena [75]. The molecular recognition events occurring between the flucorescent lectin and various carbohydrates were directly transduced to changes in the fluorescence signal [76, 77]. Finally, nanometer-sized gold particles with different ligands attached on their surface can serve as colloidal optical sensors. Color changes induced by the reversible association of lactopyranoside-functionalized gold nanoparticles in the presence of Recinus communis agglutinin (RCA120 ) lectin offer an effective method for detecting specific recognition processes [17].

8.4.2 Pathogen Detection The development of biosensors for the rapid detection of microorganisms, such as bacteria and toxins, is essential due to the undesirable health effects of pathogen infection. A monolayer of a synthetic monoalkyl disaccharide has served as a carbohydrate probe for the detection of two types of Shiga toxins produced by Escherichia coli using a quartz crystal microbalance (QCM) [78]. The sensitivity of an optical signal has a significant advantage with respect to the low detection limits required and is extensively used in the development of biosensors. Fluorene-based glycopolymers bearing mannose units have shown significant shifts in their UV–Vis absorption and fluorescent spectra upon binding to an E. coli stain [79]. The ability of glycopolythiophenes containing sialic acid or mannose moieties to detect influenza virus and

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E. coli, respectively, was evaluated by visible absorption spectrometry. Upon binding of the bacteria, the intermolecular interactions that produce a twisted polymer backbone are disrupted, and a more planar polymer backbone was obtained that resulted in a red shift in the visible adsorption of the polymer backbone [80]. 8.4.3 Specific Cell Interactions Tissue engineering has recently emerged as a new interdisciplinary field that uses cellular adhesion to artificial matrices to repair injured body parts. The adhesion of cells to extracellular matrixes is an important phenomenon required for cell growth and proliferation and depends on specific properties of the scaffold such as biocompatibility, surface energy, hydrophilicity, and the presence of cell recognition groups. The ability of certain glycopolymers to specifically recognize cells has attracted intensive scientific research for their use as cell culture matrixes. Galactose-containing polymers have been employed to induce adhesion in primary hepatocyte cells due to the specific recognition between the asialoglycoprotein receptors (ASGPR) on the surface of the cells and the galactose moieties of the polymer. The adhesion of hepatocytes to a polyLAMA-based triblock copolymer film evaporated on ethylene terephthalate plates was investigated and was found to increase with the LAMA content of the copolymer (Fig. 8.4) [8]. The interaction of hepatocyte cell lines with two different types of glycopolymers, a lactose-substituted styrene and heparin, micropatterned on glass substrates confirmed that the cells adhere specifically and strongly on the lactose-bearing polymer with little nonspecific adsorption [9]. This specific interaction of the galactose residues with the ASGPR on the hepatocyte cell membranes was employed on galactose-functionalized PS coatings on poly(l-lactic acid) (PLLA) surfaces, for their use as scaffold materials for cell culture [81]. Mouse primary hepatocyte cells have been also shown to interact specifically with glycopolymers bearing terminal glucose moieties modified at the C-6 position, in contrast to glycopolymers with glucose units modified at the other positions, which showed no significant interactions with the cells [82]. The adhesion of monocyte/macrophage cell lines was promoted on glycoconjugates of different architectures such as glycopolymers, glycocyclodextrins, and sugar-based glycoclusters possessing galactose or lactose units due to the presence of receptors with specificity for the galactose moieties on the macrophage surface [83]. Heparin-based glycopolymers were shown to selectively bind fibroblast cells

FIGURE 8.4 Schematic representation of the adhesion of hepatocyte cells onto a galactose functionalized surface.

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through the recognition of the heparin bound basic fibroblast growth factor, bFGF, by the growth factor receptors of the fibroblasts [9]. Mono and disaccharide glycopolymers containing heparin and heparan sulfate glycomimetics were found to activate the fibroblast growth factor and promote cell adhesion [84]. Cellobiose and cellotriose, two carbohydrates that rarely exist in the human body, also showed a high adhesion for fibroblast cells, even higher compared to other carbohydrate-coated surfaces, including chitobiose, maltotriose, mannotriose, and lactose, however, the mechanism of fibroblast adhesion on the cellotriose-functionalized surfaces remains unclear [85]. Recently the interactions between well-defined glycopolymer brushes covalently tethered to a substrate and cell lines were investigated. Surface-anchored glycopolymers prepared by controlled/living polymerization techniques have well-defined macromolecular architectures and high grafting densities. Their flexible nature allows the reorganization of the chains upon adhesion of cells, thus making them an interesting model as scaffolds for cell growth. Glycopolymer chains containing glucose moieties grafted onto titanium substrates were shown to minimize nonspecific protein adsorption, whereas the covalent attachment of an adhesion peptide sequence onto the glycopolymer chain ends promoted the deposition of well-spread osteoblast cells [65]. The preservation of cell function and the control over nonspecific versus specific cell adhesion on scaffold materials is very important for various applications such as in bioartificial organ devices, in molecular separation, in targeting, and in sensing. For instance, a bioartificial liver device can act as a support for patients who have suffered liver injury until the regeneration of their own hepatocyte cells. Carbohydrate-functioanlized 2-methacryloyloxyethyl phosphorylcholine polymers on PET polymeric membranes can act as biomembrane mimetic surfaces to prevent nonspecific cell function and enhance specific ligand–receptor interactions [8]. 8.4.4 Model Systems to Study Biomolecular Processes Cell surface carbohydrates such as polysaccharides, glycoproteins, glycolipids, and other glycoconjugates participate in a variety of biological functions mediated by protein–carbohydrate interactions. They provide anchors for intercellular adhesion or serve as receptors for proteins or infectious bacteria, viruses, and toxins and control the generation of biological functions [86]. Due to the importance of saccharides in biological processes, understanding the mechanism by which the carbohydrate–protein interact has become a major field of research. However, the detailed mechanism is still not well understood and remains controversial, due to the weak attractive forces involved in the binding events. Natural carbohydrate-binding proteins are typically clustered into higher oligomeric arrangements, while the saccharides protein receptors are arranged so that multiple binding events can occur simultaneously. It has thus become increasingly accepted that the specific carbohydrate interactions are achieved by polyvalency, also known as the glycoside cluster effect, which relies on aggregation with the polyvalent carbohydrates binding to lectins mainly by crosslinking [18, 87]. Chemical synthesis has provided access to multivalent sugar-based materials (glycodendrimers, glycoclusters, glycopolymers, and glycoproteins) with well-defined

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structures that allow the simultaneous multiple binding to specific proteins and can be employed to help elucidate the molecular recognition functions of saccharides [54]. It has been suggested that the shape, flexibility, molecular structure, number of binding groups, as well as the distribution of the sugar recognition units in a given glycoconjugate can greatly influence the binding events. Binding can be controlled by changing the saccharide residues or by altering their spacing [88]. However, in solution, the entanglements between the glycopolymeric chains lower their protein recognition properties [50]. The immobilization of glycopolymer chains on a surface, in close proximity to one another, forces the polymers to adopt a stretched conformation and to avoid overlapping. Therefore, immobilized carbohydrates can facilitate the analysis of protein binding and can be useful for extracting information about the cell surface topology of receptors. The surface coverage of active carbohydrates attached onto a surface has been shown to play an important role in protein binding. Thiol-modified mannose and galactose monosaccharides were shown to bind strongly to specific lectins, Con A, and jacalin, when the carbohydrate coverage was higher, which allowed the lectin to interact simultaneously with multiple sugars on the surface. However, a plateau in the amount of lectin adsorbed on the surface is reached, indicating that there is a carbohydrate density limit beyond which protein adsorption does not increase further, due to limitations on the carbohydrate binding site density [57]. Similarly, the affinities of Con A and RCA120 lectins for glucose- and galactose-based polymer brushes, respectively, determined by surface plasmon resonance (SPR), were much stronger compared to those with isolated sugar molecules [12, 64] (Fig. 8.5). Polymer brushes composed of a disulfide polymer carrying mannose residues grafted onto colloidal gold particles immobilized on glass substrates showed a greater binding affinity to Con A compared to small sugar moieties [14].

FIGURE 8.5 SPR curves of a grafted polyLAMA film on a gold sensor chip and of the RCA120 adsorption on the grafted polyLAMA layer at different concentrations.

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Glycodendrimers are highly ordered structures that can expose on their surface a different number of sugar groups and are therefore very useful tools in the systematic study of multivalency in carbohydrate–protein interactions. Starburst PMMA dendrimers possessing mannose residues exhibit a 400-fold increase in specific lectin binding compared to the monomeric form [13]. There is also considerable interest in how the size and the density of saccharides of these synthetic multivalent conjugates can influence the binding of specific proteins. For small dendrimers only monovalent interactions with lectins are expected, while large dendrimers can bind simultaneously to multiple binding sites on the lectin because they can span the distance between the carbohydrate binding sites on the protein [56]. Galactosidebased PMMA starburst dendrimers were shown to increase their binding capacity for plant agglutinin with progressive core branching and thus with increasing the number of surface-exposed sugar units [89]. Similarly, a significant enhancement of Con A binding was observed with the larger mannose-functionalized dendrimers compared to the smaller ones [19]. Control over the number of sugar moieties present on the surface of the dendrimers is particularly advantageous and can provide further information regarding the multivalent binding of the protein. To this end, PAMAM dendrimers of different generations were functionalized with mannose and hydroxyl surface groups, which allow the precise control of the sugar density. A significant increase in the binding activity was observed as the number of mannose residues on the dendrimer was increased and reached a maximum activity at about 50% sugar loading for all the dendrimer generations. As the number of sugar units increased further, a decrease in activity was observed that was attributed to unfavorable steric interactions between the lectin and the dendrimer [56]. Finally, oligosaccharide chains present on the surface of cells as part of glycoproteins or glycolipids have been shown to interact with complementary oligosaccharides leading to carbohydrate–carbohydrate complexes. The interactions between glycosphingolipids monolayers and glycoconjugate polystyrenes bearing N-glycosides of lactose, cellobiose, or gangliotriaosylceramide trisaccharide residues were investigated by SPR. The PS bearing lactose and cellobiose units did not adsorbed strongly or selectively on the glycolipid monolayer, while strong and selective interactions between the glycolipid monolayer and glycoconjugate PS bearing the trisaccharide residues were revealed and were attributed to complementary carbohydrate–carbohydrate interactions. It should be noted that the monomeric sugar molecule did not adsorb on the glycolipid monolayer [90]. 8.4.5 Inhibitors of Viruses and Toxins The nonspecific adsorption of pathogen, bacteria, and cells onto various surfaces is driven by interactions between cell proteins and the surface, lacking specific receptor-recognition binding events [91]. The nonspecific adsorption has undesirable health effects and is also linked to unwanted phenomena in the underwater parts of ships. The elimination of nonspecific binding of pathogens and cells on saccharide-coated surfaces can be employed for inhibiting the binding of pathogens on immobilized multivalent glycosilated materials that were found to be more

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effective than the monovalent ligands. For instance, N-acetyl neuraminic acid substituted dendrimers were found to be 50,000-fold more efficient as inhibitors of sendai virus compared to the monovalent monomer [92], while N-acetylglucosamine based dendrimers showed an enhanced inhibiting potency toward lectin-porcine stomach mucin compared to the monomeric sugar form. The increased inhibitory activity of the higher valence dendrimer suggests that indeed multivalency is critical [93]. The architecture of the glycoconjugates plays also an important role in their inhibitory activity. The introduction of sugar molecules onto natural proteins is an established method to produce multivalent molecules that are monodisperse and stereoregular. Helical ␣-polypeptides possessing multivalent saccharide ligands with well-defined spacing between adjacent carbohydrate sites showed inhibition of cholera toxin. When the distance between the adjacent saccharide units matched the spacing between carbohydrate binding sites of the target lectin an enhancement of up to 340-fold relative to monovalent galactose moieties was observed. Moreover, ␣-helical glycopolypeptides exhibit a greater enhancement in toxin inhibition compared to random-coil glycopolypeptides [94]. A pentacyclen core presenting galactose units was found to be an effective inhibiting ligand for the heat labile enterotoxin (LT-1) from E. coli. It was proposed that the pentagonal arrangement of the subunits is used for binding to the carbohydrate residues of gangliosides. Finally, by appropriate control over the galactose surface activity an inhibitor of LT-1, which is 105 -fold more potent compared to the monovalent galactose derivative, has been identified [95].

8.5 CONCLUSIONS AND FUTURE TRENDS This chapter provided insight into the synthesis and characterization of synthetic glycosurfaces, derived from the immobilization of small sugar molecules or more complex sugar-bearing macromolecules on a variety of supports by the grafting-to or grafting-from methods. The importance of using different analytical techniques, including spectroscopic, microscopic, and optical methods, for the characterization of the glycosurfaces has been highlighted. Advances in surface functionalization methods provide increasingly sophisticated glycosylated materials that are important for various applications, including scaffolds for cell growth, biosensors for protein and pathogen detection, nonfouling surfaces for the inhibition of the growth of viruses and toxins, and model systems to study and understand the carbohydrate–protein and carbohydrate–carbohydrate interactions. In order to synthesize glycosurfaces that possess specific functions, comparable to those of naturally occurring polysaccharides, elaborate techniques that allow the simultaneous control of the material molecular structure, stereoregularity, chain conformation, and sugar density are required. Modern techniques, presented in this chapter, have provided homogeneous and structurally defined sugar-based materials that often mimic successfully some of the basic functions of natural occurring carbohydrates such as protein and cell binding by the saccharide cluster effect, however, their synthesis is still in its early stages. Moreover, the development and use of glycosylated surfaces in commercial applications has been rarely explored, despite the

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potential that these materials hold for the future. There is a necessity for the tethering of carbohydrates on the surface of nanosized objects that could find application in areas such as nanomedicine and sensor technologies, whereas the continuing evolution of polymer brush synthesis could create innovative detection and purification platforms for biologically active molecules. Furthermore, understanding the mechanism of the recognition processes in which glycosaccharides present on the surface of the cells are involved may play an important role in the development of anticancer and antimicrobial therapies. Toward this end, the development of new synthetic strategies and advanced surface characterization tools have great potential in the realization of novel glycosurfaces and their exploitation in the emerging field of nanotechnology.

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CHAPTER 9

CARBOHYDRATE-DERIVED HYDROGELS AND MICROGELS MITSUHIRO EBARA Smart Biomaterials Group, Biomaterials Center, National Institute for Materials Science, Tsukuba, Japan

9.1 9.2 9.3 9.4 9.5 9.6

Introduction Synthesis of Hydrogels Synthesis of Micro- or Nanogels Characterizations Biological Applications Conclusions and Future Trends References

337 338 342 344 346 349 349

9.1 INTRODUCTION Hydrogels are formed with a three-dimensional (3D) network of polymer chains, where some parts are solvated by water molecules and other parts are chemically or physically linked with each other. This structure gives the interesting property that they swell but do not dissolve in an aqueous environment. Therefore, hydrogels can come from a crosslinked network of hydrophilic polymers in water, as the meaning of the prefix hydro is “aqueous,” and they maintain their 3D structure after absorbing large amounts water and swelling. 3D hydrogels based on naturally occurring polymers such as carbohydrate polymers have many advantageous features, including low toxicity and good biocompatibility because their chemical structures are similar to the bioactive glycosaminoglycan (GAG) molecules (e.g., heparin sulfate, chondroitin sulfate, hyaluronan) present in the native extracellular matrix (ECM). Furthermore, carbohydrate polymers are readily accessible at low cost. This chapter will focus

Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

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on carbohydrate-derived hydrogels and micro/nanogels as biomedical materials, and alginate, chitosan, and hyaluronic acid hydrogels, in particular, will be reviewed.

9.2 SYNTHESIS OF HYDROGELS Crosslinking of carbohydrate polymers can be established in different ways as shown below. It can be achieved by both chemical and physical crosslinking. In physically crosslinked hydrogels, the use of crosslinking agents is avoided, which might be advantageous for a great number of pharmaceutical and biomedical applications. Physical crosslinking in hydrogels can be established by, for example, ionic interactions or hydrophobic interactions. Alginate gels in which crosslinking of the anionic copolymer of 1,4-linked-␤-d-mannuronic acid and ␤-l-gluconic acid is established by CaCl2 is a well-known example [1]. Alternatively, CaSO4 or CaCO3 is used as a crosslinking agent and Na2 HPO4 as a crosslinking retardation agent because the major disadvantages of this system are that the gelation rate is too fast and hard to control [2]. The gelation rate can be easily controlled by adjusting CaSo4 /Na2HPO4 ratio in the solution. Alginate gel is also formed by gelation with polycations such as polylysine [3]. Ionic interaction is also formed by mixing negatively charged microspheres and positively charged microspheres. Dextran microspheres coated with anionic and cationic polymers exhibit spontaneous gelation upon mixing due to ionic complex formation between the oppositely charged microparticles [4]. Hydrophobic interactions have been also exploited to design physically crosslinked gels. Hydrophobic cholesterol-bearing pullulan forms hydrogel nanoparticles upon self-aggregation in water [5, 6]. Chitosan solutions containing glycerol-2-phosphate (␤-GP), which undergo temperature-controlled pH dependent sol–gel transition at a temperature close to 37◦ C, have recently been proposed [7, 8]. Combination of chitosan and poly(ethylene oxide) (PEO) also forms gel, which releases bovine serum albumin (BSA) over 70 h [9]. This type of gel-forming polymer is recently becoming increasingly attractive as an injectable hydrogel for the development of therapeutic implants. Hydrogel Classification Based on Preparation Methods Physically crosslinked hydrogels

r r r r r

lonic interactions (alginate, etc.) Hydrophobic interactions (chitosan-PEO) Hydrogen bonding interactions (HA-methylcellulose, etc.) Stereocomplexation (enantiomeric latic acid, etc.) Supramolecular chemistry (inclusion complex, etc.)

Chemically crosslinked hydrogels

r Polymerization (acryloyl group, etc.) r Small-molecule crosslinking (glutaraldehyde, etc.) r Polymer–polymer crosslinking (condensation reaction, etc.)

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339

Hydrogen-bonding interactions can also be used to formulate injectable hydrogels. Mixtures of two or more natural polymers can display rheological synergism, meaning that the viscoelastic properties of the polymer blends are more gel-like than those of the constituent polymers measured individually [10–12]. For example, blends of gelatin–agar, starch–carboxymethyl cellulose, and hyaluronic acid–methylcellulose form physically crosslinked gel-like structures that are injectable. Such blends generally exhibit excellent biocompatibility. However, hydrogen-bonded networks can dilute and disperse over a few hours in vivo due to an influx of water, restricting their use to relatively short-acting drug release systems. A novel hydrogel concept based on self-assembling of stereocomplex formation has been reported (Fig. 9.1). In general, stereocomplex formation occurs in, for example, poly(methyl methacrylate) (PMMA) and poly(lactic acid) (PLA). To create hydrogels crosslinked by stereocomplex formation, enantiomeric lactic acid oligomers were coupled to dextran [13]. A hydrogel was easily formed at room temperature upon dissolving each product in water and mixing the solution. One significant limitation of stereocomplexation is, however, the relatively restricted range of polymer compositions that can be used. A newer approach to form hydrogels in situ involves using specific molecular recognition motifs and/or supramolecular chemistry. The most common type of crosslinking interaction in this category is the formation of inclusion complexes between poly(alkylene oxide) and cyclodextrins (CDs). CDs are molecules that have hydrophilic surfaces but hydrophobic pockets that are geometrically compatible with poly(alkylene oxide) such as PEO. ␤-CD is used to crosslink PEO-grafted dextran [14]. While physically crosslinked hydrogels have the general advantages of forming gels without the need for chemical modification or the addition of crosslinking entities, they have limitations. It is difficult to decouple variables such as gelation time, network pore size, chemical functionalization, and degradation time, restricting the design flexibility of a physically crosslinked hydrogel because its strength is directly related to the chemical properties of the constituent gelators. In contrast, chemical crosslinking results in a network with a relatively high mechanical strength and,

L-Lactic

acid oligomer

D-Lactic

acid oligomer

Physical gelation via stereocomplexation between and D-lactic acid oligomer chains

L-

FIGURE 9.1 Self-assembled hydrogels by stereocomplex formation in aqueous solution of enantiomeric lactic acid oligomers grafted dextran.

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depending on the nature of the chemical bonds in the building blocks and the crosslinks, in relatively long degradation times. Chemical crosslinking calls for direct reaction of a linear or branched polymer with at least one difunctional, small molecular weight, crosslinking agent (e.g., diisocyanates, glutaraldehyde, etc.). This agent usually links two longer molecular weight chains through its di- or multifunctional groups (e.g., hydroxyl or amine of carbohydrate polymers). The formation of a hydrazone bond via the reaction of an aldehyde and a hydrazide facilitates rapid crosslinking of gel precursors [15]. Hyaluronic acid (HA) crosslinked by hydrazon bonds provided prolonged-duration local anesthesia [16]. Adipic acid dihydrazide-grafted HA was crosslinked through a selective reaction of bis(sulfosuccinimidyl) suberate to hydrazide group [17]. Chemical crosslinking has been also achieved by stabilization of HA and cellulose derivatives through the difunctional crosslinker divinyl sulfone [18]. Michael addition between a nucleophile (i.e., an amine or a thiol) and a vinyl group is another widely investigated crosslinking chemistry. As an example, a mixture of thiol-modified heparin and thiol-modified HA can be gelled with PEO diacrylate to form a hydrogel that can prolong the release of basic fibroblast growth factor [19]. Hyaluronic acid has been also enzymatically crosslinked using horseradish peroxidase and hydrogen peroxide as crosslinkers. Phenol moieties in HA-tyramine (Ty) conjugates are coupled by an enzyme-catalyzed oxidation reaction (Fig. 9.2) [20]. Dextran-Ty hydrogels can be also prepared using the same protocol [21]. Periodate-oxidized sodium alginate can be rapidly crosslinked with gelatin in the presence of sodium tetraborate to generate hydrogels [22]. Condensation reaction is also a very efficient method to crosslink polysaccharides with amine bonds. Alginate and PED-diamines were crosslined using N-ethyl carbodiimide [23]. Chemical crosslinked hydrogels can be also obtained by derivatization of a polymer with acryloyl groups followed by radical polymerization after the addition

HRP/H2O2

OH

OH

OH

OH

OH OH O OH

FIGURE 9.2 In situ gel forming of tyramine (Ty)-conjugated polymers by an enzymecatalyzed oxidation reaction.

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of an initiator system. (Meth) acrylic groups have been introduced in mono- and disaccharides, which can be used for synthesis of hydrogels. A hydrogel is formed after the addition of an initiator system containing of N,N,N ,N -tetramethylenediamine (TEMED) and ammonium peroxydisulfate (APS) to an aqueous solution of the methacryl–dextran (MA–dextran) containing N,N -methylene-bis-acrylamide (MBAAm). A polymerizable dextran derivative is also obtained by the reaction of dextran with maleic anhydride. Sucrose was reacted with glycidyl acrylate to introduce double bonds. Since the reaction mixture contained sucrose modified with more than one double bond, its polymerization resulted in a crosslinked network of sucrose hydrogels (sucrogels). Superporous sucrogels were also prepared by adding a gas-forming agent during polymerization. Chitosan undergoes crosslinking by radical induced polymerization in presence of potassium persulfate at 60◦ C, leading to extensive crosslinking of fragmented chains on subsequent cooling at 4◦ C. Photocrosslinked hydrogels have recently gained increased attention in biomedical applications because aqueous macromer solutions containing cells and/or bioactive factors can be delivered in a minimally invasive manner and then rapidly crosslinked in physiological conditions in situ following brief exposure to ultraviolet (UV) light. Following exposure to UV or visible light, chemicals called photoinitiators generate free radicals that can initiate the polymerization process. The free radicals that are created convert aqueous macromer solutions into hydrogels. Photocrosslinkable alginate macromers, for example, were photocrosslinked using UV light with photoinitiator [24]. Dextran hydrogels were also photopolymerized in the presence of a photoinitiator. The hydroxyl groups of native dextran were converted to acrylate groups to make hydrogel precursors with different substitution degrees [25]. Ionizing radiation crosslinking that utilizes electron beams, gamma rays, or X rays also produces a crosslinked structure via free radical reactions. A series of excellent polyvinyl alcohol (PVA)/starch blend hydrogels were prepared by gamma and electron beam radiation at room temperature [26]. To combine the advantages of synthetic and carbohydrate polymers, interpenetrating polymer network (IPN) type of hydrogels have been prepared. An IPN is formed when a second hydrogel network is polymerized within prepolymerized hydrogels. For example, a mixture solution of chitosan and synthetic 2-hydroxymethyl methacrylate (HEMA) was crosslinked by glutaraldehyde in aqueous solution to form IPN hydrogel [27]. IPN hydrogels based on chitosan with N-vinylpyrrolidinone (NVP) as well as its copolymer with HEMA were also synthesized using the photopolymerization technique [28]. The combination of konjac glucomannan (KGM) and poly(acrylic acid) (PAA) has been used as an enzymatic degradable and pH-sensitive IPN can be synthesized using crosslinking PAA by MBAAm with konjac glucomannan (KGM [29]). A series of semi-IPN materials have been prepared by blending polymerization of acrylic acid (AA) in cationic starch (CS) and poly(methacryloyloxyethyl trimethylammonium chloride) (PDMC) solution [30]. Thermoresponsive guar gum (GG)/poly(N-isopropylacrylamide) (PNIPAAm) hydrogels with IPN were synthesized [31]. The thermoresponsive GG/PNIPAAm IPN hydrogels with different response rates can be prepared by modifying the proportion of GG to PNIPAAm. Compared with pure PNIPAAm hydrogels, GG/PNIPAAm hydrogels with reversible

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thermoresponsive characteristics exhibited faster deswelling rates and lower water retentions at low GG content (below 15 wt%). The introduction of GG component with IPN technology could improve the temperature sensitivity and permeability of GG/PNIPAAm IPN hydrogels, which could be expected as good candidates for the controlled drug delivery system with both thermoresponsive and specific-colonic drug release behaviors. Electrospinning has also been used to design hydrogels with applicability in biomedical fields [32]. The number of recent studies regarding electrospinning polysaccharides and their derivatives is increasing dramatically. Various polysaccharides such as alginate, cellulose, chitin, chitosan, HA, starch, dextran, and heparin have potential to be used for electrospinning.

9.3 SYNTHESIS OF MICRO- OR NANOGELS The pursuit for targeted drug delivery systems has led to the development of highly improved biomaterials with enhanced biocompatibility and biodegradability properties. Micro- and nanoscale components of hydrogels prepared from carbohydrates have been gaining significant importance due to their potential uses in cell-based therapies, tissue engineering, cancer therapy, and drug delivery. In this section, some of the recent methodologies used in the preparation of carbohydrate-derived micro/nanogels have been discussed. Microgel particles are synthesized by the following most commonly used methods: emulsion polymerization, inverse emulsion polymerization, or via methods involving the use of radiation. Emulsion polymerization is a versatile technique that yields narrow particle size distributions. Emulsion polymerization can be performed in the presence of added surfactant or in the absence of added surfactant, also called precipitation polymerization. Microgels of chitosan and Pluronic F127 were prepared by the emulsion crosslinking method employing glutaraldehyde as a crosslinker [33]. An anticancer drug was easily encapsulated into hydrogels. Protein-loaded dextran microspheres were prepared by a water-in-water emulsion technique [34–37]. With this technique, an aqueous solution of methacrylated dextran (MA–dextran) is emulsified in an aquesou solution of PEO. Subsequently, the dispersed MA–dextran phase is crosslinked by radical polymerization of the dextran-bound methacryloyl groups. This method renders microspheres with a hydrogel character of which the crosslink density can be controlled by the water content and the degree of substitution of the MA–dextran. Thioridazine-containing ethyl cellulose (EC) microcapsules were prepared in the presence of gold nanoparticles via the water/oil/water (W/O/W) emulsification solvent–evaporation method [38]. Polyacrylamide-grafted guar gum was also prepared by the emulsification method [39]. Microgels consisting of dextran have been prepared by UV polymerization. Dextran and Concanavalin A (Con A) precursors were modified with acrylic side groups and then UV polymerized to produce covalently bonded mixtures [40]. Insulin was released from the microgels when there was a high concentration of glucose present.

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A novel method has been also reported on preparing nanogels [41]. The method involves the Maillard dry-heat process and heat gelation process. First, dextran–lysozyme conjugates were produced through the Maillard reaction. Then, the conjugate solution was heated above the denaturation temperature of lysozyme to produce nanogels with a lysozyme core and dextran shell. The nanogels are of spherical shape having a hydrodynamic diameter of about 200 nm. Chitosan nanogels were also produced by a convenient method based on electrostatic attractions. Chitosan and ovalumn solutions were mixed and then the chitosan chains were partly trapped in the nanogel core upon heating because of the electrostatic attraction between chitosan and ovalbumin, while the rest of the chitosan chains should form the shell of the nanogels [42]. Recently, a solution of N-[(2-hydroxy-3-trimethylammonium)propyl]-modified chitosan was combined with sodium tripolyphosphate (TPP) in order to prepare the microgels by ionic crosslinking. Self-organizing nanogels have also been studied. Pullulan, a polysaccharide consisting of maltotriose units, has been combined with cholesterol to form selfaggregating hydrogels (Fig. 9.3) [43–46]. The cholesterol groups allow crosslinking within the gel to occur. Also, cholesteryl-group-bearing pullulan was shown to bind to hydrophobic substances and proteins. These findings display promise for the use of self-aggregating nanogels in medicine because the hydrophobic polysaccharide is able to complex with other molecules, helping to stabilize them against changes in the external environment. Cationic nanogels composed of cholesteryl-group-bearing pullulans functionalized with an ethylenediamine group stabilizes proteins [47]. Pullulan–deoxycholic acid conjugates have also been synthesized and histidine was further conjugated to assemble into nanogels [48].

FIGURE 9.3 Self-aggregated nanoparticles of cholesteryl-bearing pullulan.

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9.4 CHARACTERIZATIONS While hydroegls have a wide range of applications, their basic characteristics exhibit common physicochemical origins. For example, the weak interaction between their structural elements is one obvious key feature. The crosslink density of hydrogels is also one of the important factors to determine a wide variety of important properties, including the swelling, transport/release kinetics, and mechanical properties. Although numerous macroscopic studies were performed with various hydrogels over the past years, there is still a growing need for a detailed understanding of the properties of hydrogels at a microscopic and nanoscopic scale. Scanning electron microscopy (SEM) is widely used in the structural research of hydrogels. SEM provides information about the micromorphology and spatial structure of hydrogels [49]. For example, the interior morphology of dextran hydrogels was visually examined using SEM and reliable quantitative data of pore characteristics was obtained. The swollen dextran hydrogel showed a different pore size and morphology at the surface and the interior. Since about two decades, environmental scanning electron microscope (ESEM) allows wet matter to be studied at low vacuum (up to 10 torr) of the operator’s choice. ESEM should be a suitable choice for studying the structure of water-swollen gels [50]. Atomic force microscopy (AFM) images can provide a smooth gel surface with nanometer-sized protrusions. The AFM images provide quantitative topographic information: for example, the height of the roundish protrusions varied from 5 to 15 nm [51]. However, AFM is restricted to height variations less than 10 ␮m and a scanning field smaller than 100 × 100 ␮m2 . AFM is also perfectly suited to determined local mechanical properties such as Young’s modulus as it uses a force sensor to apply and accurately measure forces on the submicrometer scale. Local elastic properties of any sample surface can be obtained under ambient conditions in air and in water with high precision. In principle, these properties are revealed by force curves, showing the indentation of the surface as the probe loads the sample. The above-mentioned examples demonstrate how different microscopy methods complement the exploration of the swelling hydrogels. Since the softness of hydrogels comes from a crosslinked three-dimensional network capable of absorbing solvent to a high degree without dissolution, the swelling studies are important to understand water uptake of hydrogels as well as drug release characteristics through the gels. It depends upon the nature and extent of interaction between solvent molecules and polymer chains in addition to porosity of hydorgels and nature of hydrophilic groups on the polymer [52, 53]. The swelling ratio is usually determined by immersing the dry hydrogels in aqueous solutions of the desired conditions [54,55]. After regular periods of time, they are removed from the aqueous solution and the removal of excess surface water and weighed. The swelling ratio is calculated from the equation: Swelling ratio = (Ws − Wd )/(Wd ), where Ws and Wd represent the weights of the swollen and dry-state samples, respectively. The interaction of water with such polymers and its distribution within the polymeric system are critical for the hydrogel’s mechanical strength and also their ability

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to control drug release. The rate at which water diffuses into the hydrophilic matrix and forms the barrier gel layer, and the subsequent diffusion of water through this gel layer, are major factors determining the drug release rate from such devices. Drug release is further influenced by the dynamics of the polymer chains and the polymer mesh size, which represent a barrier for drug diffusion from the polymer matrix. Detailed characterization of the gel layer and, more specifically, of the types of water that exist within the gel, are fundamental to the optimization and prediction of drug release from swollen tablets. Three types of water have been classified in hydrophilic polymer gels: nonfreezing or bound water, freezing interfacial or intermediate water, and free water. Many methods can be used to determine and distinguish the different states of water. The most useful are nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). NMR spectroscopy is a powerful technique for detailed studies of the structure, mobility, and hydration properties of various polymeric systems [56, 57]. For example, different types of water can be distinguished in cellulose-based hydrogels. In particular, the fraction of free water in hydrated cellulose ethers is important for predicting rates of drug release from the swollen tablets. Different states of water in hydrogel matrices can be specified by measuring the water proton 1 H-NMR spin-lattice (T 1 ) and spin–spin (T 2 ) relaxation times. Fourier transform infrared (FTIR) is used to confirm the modification degree of hydrogels. For example, the IR spectrum of chitosan shows signals at 1647 and 1590 cm−1 for the C–O stretching (amide) and N–H bending (amine), respectively. The spectrum of carboxymethylchitosan, however, shows a new peak appears at 1718 cm−1 , which is assigned to the carbonyl groups on the side chains [58]. Introduction of acrylic groups on dextran was also confirmed by FTIR spectrum, which shows the emergence of new bands at 813 cm−1 for vinylidene deformation [41]. The crosslinking reaction and the degree are also confirmed by FTIR. The IR spectrum of crosslinked carboxymethylcellulose shows an intense peak centered at 1575 and 1540 cm−1 . These two peaks can be related to the presence of the crosslinking amidic groups [59]. The crosslinking degree is also determined by potentiometric titration. Potentiometric titration is carried out to verify the number of carboxylate groups of the polysaccharide involved in the crosslinking reaction. One of the most important aspect of characterization of gels seems to clarify their backbone dynamics together with conformations as viewed from their highly heterogeneous nature. As a first step to this end, backbone dynamics of gel network can be very conveniently characterized by means of simple comparative high-resolution 13 C-NMR measurements. The dynamic feature of such gels can be related with the revealed network structures as viewed from various types of spin–relaxation processes. The interaction of the fluid medium water with the polymeric matrices was investigated by NMR relaxation times at low resolution [60]. On the other hand, lowresolution NMR studies allow the determination of the water transport properties in the hydrogels. The interaction of the fluid medium water with the polymeric matrices is investigated by NMR relaxation times. The viscoelastic property is also one of the important characteristics of hydrogels. The viscoelastic properties of dextran gels, for example, are measured in situ with

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FIGURE 9.4 In situ rheometric measurements of hydrogel formation from acrylatemodified dextran macromers.

rheometric measurement. The elastic component of the modulus increases sharply as gelation occurred (Fig. 9.4) [59, 61]. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies are usuful for understanding the crystalline nature of the drug after encapsulation into hydrogels [62].

9.5 BIOLOGICAL APPLICATIONS Water-swollen crosslinked hydrogels have varied applications in fields such as pharmaceuticals and biomedicines. Among these applications, hydrogel-based drug delivery devices have become a major area of study, and several commercially available products are already in the market. Proteins, peptides, DNA-based drugs can be delivered via hydrogel carrier. The various properties of hydrogels such as biocompatibility, hydrophilicity, and flexibility all make it ideal for use as a drug delivery matrix. The colonic region, for example, has been considered as a possible absorption site for orally administered proteins and peptides, mostly due to a lower proteolytic activity in comparison to that in the small intestine. Several gels are currently being investigated as potential devices for colon-specific drug delivery. These include chemically or physically crosslinked dextran or guar gum, for example. They are designed to be highly swollen or degraded in the presence of colonic enzymes or microflora, providing colon specificity in drug delivery. Ionically crosslinked chitosan gels have demonstrated encapsulation of doxorubicin and shown good in vitro cytotoxicity. A hydrophilic drug, propranolol hydrochloride, was also encapsulated in gellan gum beads in an aqueous environment by the ionotropic gelation method [63].

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Hydrophobic modification of chitosan, dextran, and pullulan forms monodisperse hydrogel nanoparticles of 20–30 nm via physical crosslinking [5]. The hydrophobic antitumor drug adriamicin (ADR) is easily encapsulated by simply mixing the pullulan suspension with ADR. Various proteins such as chymotrypsin, BSA, and insulin can be also incorporated into the nanoparticles. Rapid complexation was observed for insulin within 10 min after mixing with pullulan. The resulting microenvironment protected the protein effectively against the thermal denaturation aggregation and enzymatic degradation. Microgels consisting of dextran derivatives crosslinked with the lectin Concanavalin A (Con A) have been incorporated with insulin [40, 64]. It was observed that the insulin was released in higher quantities when there was a high concentration of glucose present because the dextran and glucose compete for binding sites with Con A, which tears the gel and releases the insulin. Recently, a solution of modified chitosan, N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC), was combined with sodium tripolyphosphate (TPP) in order to prepare the microgels and methotrexate disodium (MTX), a cytotoxic drug utilized in the treatment of cancer, was loaded into the gel [65]. Further, the gels were conjugated with apo-transferrin because this protein is able to enter cells through receptor-mediated endocytosis. During this process, the hydrogels undergo a lowering of their pH from 7.4 to 5.0 within the cell, which enhances the speed at which the MTX would be released into the lower pH environment in the tumor cells. Cationic nanogels composed of cholesteryl-group-bearing pullulans functionalized with an ethylenediamine group (CHPNH2 ), which are amphiphilic polysaccharides, are one such material being synthesized for utilization in protein delivery [47]. Proteins such as bovine serum albumin and ␤-galactosidase were encapsulated within the nanogels, and it was found that once the nanogel particle was taken up by the cell, the nanogel–protein complex dissociated and the protein was released. Injectable hydrogels have been also applied in biomedical fields since they can be used as scaffolds, drug and gene carriers, and the like. In-situ-forming hydrogels triggered by environmental stimuli have emerged as a promising injectable strategy. N-palmitonyl chitosan undergoes the pH-triggered hydrogelation after being injected through a needle into the subcutaneous space of a rat model (Fig. 9.5) [66]. A calcium crosslinked alginate can be delivered effectively into the infracted myocardium by intracoronary injection to prevent left ventricular remodeling early after myocardial infarction (MI). A calcium crosslinked alginate solution undergoes liquid-to-gel phase transition after deposition in infracted myocardium [67]. Examination of hearts harvested after injection showed that the alginate crossed the infarcted leaky vessels and was deposited as hydrogel in the infarcted tissue. Post-mortem analysis showed that the biomaterial increased scar thickness by 53% compared with control and was replaced by myofibroblasts and collagen. Since the chemical structures of polysaccharides are similar to the bioactive GAG molecules present in the ECM, they are recently used in the new field of tissue engineering as scaffolds for repairing and regenerating a wide variety of tissue and organs. One of these applications of alginate hydrogels, for example, is to engineer cartilage tissue in vivo to serve as a bulking tissue in the treatment of reflux of fluid from bladder to kidney via a minimally invasive surgical procedure. Alginate

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FIGURE 9.5 Schematic illustrations of pH-triggered hydrogelation of aqueus N-palmitoyl modified chitosan injected through a needle into subcutaneous space of a rat.

hydrogels have also been used to transplant chondrocytes and regenerate new tissues such as bone tissue, blood vessels, nerves, and livers. Incorporation of appropriate adhesion factors, growth factors, or components of the ECM into the gels may allow one to manipulate the cell function and tissue formation. Chitosan hydrogels have been also utilized to engineer cartilage, bone, and nerve tissue via supplementation with growth factors or cell adhesive molecules. In particular, the combined use of chitosan hydrogels with bioactive inorganic particles or synthetic polymers has been widely attempted to optimize the properties of these hydrogels for tissue engineering. The possible toxicity of hydrogels is an important issue to be considered particularly with respect to biomedical applications. Schmidt and co-workers synthesized biomimetic hydrogels in order to promote tissue repair using hyaluronic acid (HA) as starting material [68]. They prepared a variety of glycidyl methacrylate-HA (GMHA) conjugates, which were then photopolymerized to form crosslinked GMHA hydrogels. Upon conducting degradation studies, they observed that a range of degradation rates could be attained. Increased amounts of conjugated methacrylate groups corresponded to increased crosslink densities and decreased degradation rates. However, no significant effect on human aortic endothelial cell cytocompatibility and proliferation was observed. Rat subcutaneous implants of the GMHA hydrogels showed good biocompatibility, very low inflammatory response, and similar levels of vascularization at the implant edge compared with those of fibrin-positive controls. Their results indicate that such GMHA hydrogels can potentially be used in a variety of woundhealing applications. Thus, in general, by systematically tuning the composition and by performing specific chemical modifications, a variety of biocompatible hydrogels can be synthesized and can potentially be used in a wide range of applications such

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as sustained, targeted drug delivery and tissue engineering. They are used not only in generic drug delivery systems but also in anticancer therapies, treatments for diabetes, protein delivery systems, biosensor applications, microlens development, and tissue regeneration.

9.6 CONCLUSIONS AND FUTURE TRENDS Novel strategies for the design and synthesis of highly efficient, biocompatible hydrogels will facilitate the creation of new classes of biomaterials for drug delivery and tissue engineering. While most hydrogels are capable of releasing drugs either continuously or intermittently, having accurate, desired release rates would be a major improvement. The use of hydrogel delivery systems for the development of clinically viable products, using recombinant proteins requires further development before these systems can be used without further damaging the proteins. In addition to soft-tissue engineering and drug delivery, bone replacement therapy is another area where hydrogels might find plausible applications. Since the chemical structures of carbohydrate hydrogels are similar to the ECM proteins, they are extremely crucial therapeutic materials in relation to drug delivery, biosensors, and tissue engineering applications due to their biocompatibility, biodegradability to nontoxic products within the body, and swelling capacities. More recently, micro- and nanoscale hydrogels have become increasingly popular due of their ability to target areas.

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40. Tanna, S., Taylor, M. J., Sahota, T. S., and Sawicka, K. (2006). Glucose-Responsive UV Polymerised Dextran–Concanavalin A Acrylic Derivatised Mixtures for Closed-Loop Insulin Delivery. Biomaterials 27, 1586–1597. 41. Li, J., Yu, S., Yao, P., and Jiang, M. (2008). Lysozyme-Dextran Core-Shell Nanogels Prepared via a Green Process. Langmuir 24, 3486–3492. 42. Yu, S., Hu., J., Pan, X., Yao, P., and Jiang, M. (2006). Stable and pH-Sensitive Nanogels Prepared by Self-Assembly of Chitosan and Ovalbumin. Langmuir 22, 2754–2759. 43. Akiyoshi, K., Nishikawa, T., Mitsui, Y., Miyata, T., Kodama, M., and Sunamoto, J. (1996). Self-Assembly of Polymer Amphiphiles: Thermodynamics of Complexation between Bovine Serum Albumin and Self-Aggregate of Cholesterol-Bearing Pullulan. Colloids Surf. A 112, 91–95. 44. Akiyoshi, K., Nishikawa, T., Shichibe, S., and Sunamoto, J. (1995). Stabilization of Insulin upon Supramolecular Complexation with Hydrophobized Polysaccharide Nanoparticle. Chem. Lett. 24, 707–708. 45. Nishikawa, T., Akiyoshi, K., and Sunamoto, J. (1996). Macromolecular Complexation between Bovine Serum Albumin and the Self-Assembled Hydrogel Nanoparticle of Hydrophobized Polysaccharides. J. Am. Chem. Soc. 118, 6110–6115. 46. Nishikawa, T., Akiyoshi, K., and Sunamoto, J. (1994). Supramolecular Assembly between Nanoparticles of Hydrophobized Polysaccharide and Soluble Protein Complexation between the Self-Aggregate of Cholesterol-Bearing Pullulan and alpha-Chymotrypsin. Macromolecules 27, 7654–7659. 47. Ayame, H., Morimoto, N., and Akiyoshi, K. (2008). Self-Assembled Cationic Nanogels for Intracellular Protein Delivery. Bioconjug. Chem. 19, 882–890. 48. Na, K., Lee, E. S., and Bae, Y. H. (2007). Self-Organized Nanogels Responding to Tumor Extracellular pH: pH-Dependent Drug Release and in vitro Cytotoxicity against MCF-7 Cells. Bioconjug. Chem. 18, 1568–1574. 49. Kim, S.-H., and Chu, C.-C. (2000). Pore Structure Analysis of Swollen DextranMethacrylate Hydrogels by SEM and Mercury Intrusion Porosimetry. J. Biomed. Mat. Res. Part B 53, 258–266. 50. Donald, A. M. (2003). The Use of Environmental Scanning Electron Microscopy for Imaging Wet and Insulating Materials. Nat. Mat. 2, 511–516. 51. Weisenhorn, A. L., Khorsandi, M., Kasas, S., Gotzos, V., and Butt, H.-J. (1993). Deformation and Height Anomaly of Soft Surfaces Studied with an AFM. Nanotechnology 4, 106–113. 52. Remu˜na´ n-L´opez, C., and Bodmeier, R. (1997). Mechanical, Water Uptake and Permeability Properties of Crosslinked Chitosan Glutamate and Alginate Films. J. Controlled Release 44, 215–225. 53. Hsu, S.-H., Leu, Y.-L., Hu, J.-W., and Fang, J.-Y. (2009). Physicochemical Characterization and Drug Release of Thermosensitive Hydrogels Composed of a Hyaluronic Acid/Pluronic F127 Graft. Chem. Pharm. Bull. 57, 453–458. 54. Ebara, M., Aoyagi, T., Sakai, K., and Okano, T. (2001). The Incorporation of Carboxylate Groups into Temperature-Responsive Poly(N-Isopropylacrylamide)-Based Hydrogels Promotes Rapid Gel Shrinking. J. Polym. Sci. Part A 39, 335–342. 55. Ebara, M., Aoyagi, T., Sakai, K., and Okano, T. (2000). Introducing Reactive Carboxyl Side Chains Retains Phase Transition Temperature Sensitivity in N-Isopropylacrylamide Copolymer Gels. Macromolecules 33, 8312–8316.

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CHAPTER 10

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES ARCHANA BHAW-LUXIMON Department of Chemistry, University of Mauritius, Reduit, Mauritius ´

10.1 10.2 10.3 10.4

Introduction Principles of Controlled Drug Delivery Polysaccharides as Nanodrug Delivery Devices Modified Polysaccharides as Nanodrug Delivery Devices 10.4.1 Polysaccharide Grafts 10.5 Modified Polysaccharides—Block Systems 10.5.1 Oligosaccharide-b -Copolymer 10.5.2 Dextran-b -Copolymer 10.5.3 Amylose-b -Copolymer 10.5.4 Hyaluronan-b -Copolymer 10.6 Mode of Delivery of Modified Polysaccharide Nanoparticles 10.6.1 Oral Drug Administration 10.6.2 Nasal Administration 10.7 Conclusions and Future Trends References

355 356 357 358 358 370 371 372 377 377 379 379 385 387 387

10.1 INTRODUCTION The ability to characterize, manipulate, and organize matter systematically at the nanometer scale has revolutionized science, engineering, technology, and inevitably Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

355

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MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

drug delivery and therapeutics. Researchers are at the threshold of reaching the longcherished goal of precise delivery of drugs to a specific compartment in the target cell. Nanotechnology offers great promise of developing a diverse range of drug delivery systems encompassing recognition at the molecular level. The dream will come true when devices can reach flawless drug targeting at the cellular and subcellular levels and behave as specialized “living cells.” However, scientists from both developed and developing worlds are faced with the ethical and scientific challenge to produce and implement nanotechnologies at reasonable costs. Nanoparticles derived from renewable resources can play a significant role in lowering costs of nanotechnologies. One such family of resources is natural polysaccharides. These have been and are being modified to improve their performance as drug delivery devices with much emphasis on targeting.

10.2 PRINCIPLES OF CONTROLLED DRUG DELIVERY Conventionally, multiple daily doses of therapeutic agents in the form of tablets or capsules are required for the treatment of diseases. The drug is absorbed in the systemic circulation via a stepwise manner (Fig. 10.1). Physical or chemical microencapsulation is the mechanism utilized by 65% of all sustained-release systems, for example, Arthritis Bayer, and No Doz. The Wurster process was the first physical process developed in 1949 and involved surrounding the therapeutic agent by a coating. Chemical encapsulation is achieved as coacervation. Implantable drug delivery systems are still at the development stage. The commercially available implant systems include Norplant and pumps such as insulin pumps. They deliver the drugs directly into the bloodstream at a controlled rate. Transdermal drug delivery systems are used in the continuous administration of drug from the

Gastrointestinal tract Tablet/capsule Epithelial surf ace

Mucus lining

Adsorption

Diffusion

FIGURE 10.1 Conventional drug delivery.

POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES

Nanocapsule

Nanosphere

Dendrimer

Vesicle

357

Core–shell micelle

FIGURE 10.2 Morphologies of drug carriers.

surface of skin into the circulatory system. Transderm Scop (CIBA) was the first commercialized system releasing the anti-motion sickness drug scopolamine. Site-directed drug delivery systems include nanoparticles. Synthetic and natural polymers are the basic constituents of nanoparticles. Important factors that have to be considered for the design of such systems are bioacceptability of the polymer (biodegradable and biocompatible), physicochemical properties of the drug, and the type of therapy possessed by the drug [1–3]. Biodegradable/biocompatible polymers can be natural, modified natural, or synthetic. Natural polymers that have been used widely include polysaccharides. Nanoparticles are a collective name for nanospheres, nanocapsules, and micelles. Nanoparticle-sustained drug delivery systems offer several advantages over conventional delivery such as maintenance of optimum therapeutic concentration of drug in the blood or cell, elimination of frequent dosing, and better patient compliance. Nanosized particles can also be administered intravenously as the smallest blood capillary has a diameter of approximately 4 ␮m. Figure 10.2 illustrates the different morphologies of pharmaceutical carriers being used/tested as drug carriers. Nanoparticulate systems have become very important in the strategies considered for drug delivery. For instance, in the field of anticancer drug delivery, macromolecular anticancer drugs have been shown to remain in tumor tissue for a much longer period than in normal tissue, following both direct injections into the tumor and indirect accumulation from the bloodstream. The combination of poor tissue drainage with increased tumor vascular permeability results in a phenomenon termed the enhanced permeability and retention (EPR) effect. The effect appears to apply to all tumors so far examined, suggesting that passive accumulation of macromolecular drugs may be a universal phenomenon. Hence the rationale justifying the development of macromolecular anticancer drugs is clear. A recent trend has emerged with emphasis on the use of nanoparticle drug delivery systems for the treatment of infectious diseases such as tuberculosis, malaria, and HIV/AIDS. 10.3 POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES The design of nanodrug delivery devices requires the consideration of a number of important factors among which are toxicity and biodegradability. Polysaccharides offer the advantages of being highly stable, nontoxic, hydrophilic, and biodegradable, which are critical aspects to consider for biomedical applications. Much research on

358

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

the use of native polysaccharides as controlled delivery systems has been performed. A turning point was observed in that field due the vast possibility offered by the numerous polysaccharide OH/NH2 groups, which can be used for chemical modification [4]. In addition, OH groups also allow specific mucoadhesion or receptor recognition. Thus, natural polysaccharides have been combined with synthetic polymers to yield novel materials for biomedical applications. For instance, micelles having sugar groups on their exterior are expected to have wide utility in the field of drug delivery as glycoreceptor-directed carrier systems as it is known that RCA-1-lectin (Ricinus communis agglutinin-1 lectin) recognizes ␤-d-galactose residues. They can be very useful in cancer therapy as hepatocyte galactose receptor for organ-specific chemotherapy, for example, primary and metastatic liver cancer. One such system is the galactose-polyethyleneglycol (PEG)-b-polylactide (PLA) system developed by Kataoka et al. [5]. Natural polysaccharides are obtained from various sources such as algae, shells of crustaceans, and plants (Table 10.1). Polysaccharides of algal origin are alginates, agarose, and carrageenans, whereas polysaccharides of plant origins are starch and cellulose. Hyaluronic acid is another polysaccharide naturally present in human and animal tissues. Natural polysaccharides offer the advantages of being: obtained from renewable resources, biodegradable and biocompatible. However, associated with these, they suffer from rapid erosion and low adhesion to mucosal tissues. The presence of functional groups such as OH and NH2 has offered the possibility of chemically modifying natural polysaccharides to produce improved materials. Crosslinked chitosan was among the first polysaccharides used to prepare nanoparticles. However, due to the toxicity of the crosslinker used, namely glutaraldehyde, it had limited applications in the biomedical field. The advent of biocompatible crosslinkers such as tartaric acid revived the interests in crosslinked chitosan as nanoparticles [13]. These nanoparticles (270–370 nm) were found to be stable in acidic, neutral, and alkaline media. Agarose hydrogel nanoparticle (500 nm) has also been used as a suitable matrix for entrapment of therapeutic proteins, which are initially added to the agarose solutions before gelation [14]. The viscosity, thus the concentration of the agarose solutions, should be low (1–3%) to obtain nanoparticles. 10.4 MODIFIED POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES Polymeric micelles can have a spherical or a cylindrical shape. Most micelles used for drug targeting have a spherical shape [15]. A spherical polymeric micelle structure forms from block copolymers or graft copolymers. The next part of this chapter will deal with modified polysaccharides, namely graft and block copolymers (Fig. 10.3): synthetic approach, structures, solution properties, and drug loading. 10.4.1 Polysaccharide Grafts Polysaccharides have been modified to increase their hydrophobicity, promoting new biological activities making them more adaptable to site targeting and also to give

359

Chitosan [8a–e]

Alginates [7a]

Agarose [6a]

Polysaccharide

Repeat Unit

n

TABLE 10.1 Natural Polysaccharides as NanoDrug Delivery Devices

n

n

Shells of crustaceans and insect exoskeletons

Brown seaweeds (kelp)

Red seaweeds, e.g., Gracilaria, Gelidium

Source

(Continued)

Cyclosporine A loaded CN [8f] Insulin-loaded CN [8g] 5-Fluorouacil-loaded CN [8h]

Isoniazid, rifampicin, and pyrazinamide alginate (235.5 nm) [7b]

Ovalbumin-agarose hydrogel nanoparticle (504 nm) [6b]

Sample Nanoparticles

360 Repeat Unit

n

n

n

n

Natural Polysaccharides as NanoDrug Delivery Devices (Continued)

Hyaluronic acid [10a,b]

Carrageenan [9a]

Polysaccharide

TABLE 10.1

Human and animal tissues

Red seaweeds, e.g., Hypnea, Chondrus crispus

Source

Cisplatin-incorporated HA (100–200 nm) [10c] HA-5␤ cholanic acid conjugate (350–400 nm) [10d]

␭ carrageenan-loaded dexchlorphenoramine maleate (50 nm) [9b]

Sample Nanoparticles

361

Starch [12a] (mixture of amylase and amylopectin)

Cellulose [11a]

n

n

n

Granules in plant cells

Plants

Folate-conjugated starch nanoparticles [12b]

Cellulose nanocrystalsfluorescein-5 isothiocyanate [11b]

362

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

FIGURE 10.3 Graft and block polysaccharides self-assembly.

rise to amphiphilic systems. This has been achieved using their numerous OH/NH2 groups to attach synthetic polymers or small molecules. Three strategies have been reported so far for graft systems: (i) using native polysaccharides as macroinitiators [16–20], (ii) using protected polysaccharides as macroinitiators followed by deprotection [21–30], and (iii) end-to-end coupling reaction between preformed polymers and hydroxyl/amine group on polysaccharides [31]. The protection–deprotection approach, in spite of being tedious in some cases, provides some control on the grafting compared to direct use of polysaccharides as macroinitiators. We will review in the next part only the graft species that have shown some promise in drug loading. We will look at the choice of synthetic polymer/molecule, grafting techniques, solution properties, cytotoxicity, and drug loading. One very interesting aspect with polysaccharides is that the position of the free OH after polymer grafting can be very useful in terms of site targeting. For example, Kopecek and Duncan have reported that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing galactose linked at the C-6 position had an effective targeting property to the liver, which was proved from studies on rats [32a]. HPMA with galactosamine linked at the C-2 position also showed effective delivery of polymer-conjugated drug to the liver [32b]. Table 10.2 summarizes some graft copolymer systems that have been synthesized, their corresponding solution properties, and their possible use in drug loading.

10.4.1.1 Polysaccharide as Macroinitiators for Grafting Polysaccharides can be used directly as macroinitiators for the grafting of polymer chains. Different type of copolymerization mechanisms have been applied mainly based on ringopening polymerization (ROP) and atom transfer radical polymerization (ATRP). Cellulose is one polysaccharide that has been used with both techniques. Cellulose is the most abundant natural biomaterial in the world and presents good biocompatibility, biodegradability, and renewability. Modification of cellulose and its derivatives by graft copolymerization can provide a significant method to alter its physical and chemical properties. In an attempt to combine the advantages offered by cellulose with those of the well-known biocompatible polylactide (PLA), cellulose-g-PLAs were synthesized. Cellulose was used as a macroinitiator for ring-opening graft polymerization of l-lactide (l-LA) in an ionic liquid using Sn(Oct)2 as catalyst [41]. Apart from preferential reaction at C-6, the order of reactivity was established

MODIFIED POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES

TABLE 10.2

363

Some Drug-Loaded Graft Polysaccharide Copolymer Systems

Copolymer

Size (nm)

Dex-g-PCLn [31] Dex-g-PEO-C16 Vit12-Dex-g-PEO-C16 [33] Dex-MA-g-PNIPAAm [34] Starch-g-fatty acid [35] Lactic acid-g-chitosan [36] PLA-g-chondroitin sulfate [37] PEO-g-chitosan N-phthaloylchitosan-gPEG

45–140 14 33

Drug Loaded/ Fluorescence Label

65–75 10

— Cyclosporine A (immunosuppressant) 5–8% 10-hydroxycamptothecin 0.62% Indomethacin—16% BSA

200

Flutamide

262 100–250

All transretinoic acid [38] Methotrexate [39] Camptothecin [40]

60–300

Possible Grafting Position

CMC

C-2 C-3

— 3.8 mg/L 7.4 mg/L

C-2

<5 mmol L−1

— C-2

—

C-6, C-2, C-3 —

— — 0.07 mg/mL 28 ␮g/mL

as C6–OH > C3–OH > C2–OH, which is consistent with reported work on the reactivity of hydroxyl groups on cellulose [42]. Cytotoxicity study on the proliferation of 3T3 fibroblast showed no inhibition at concentration below 700 mg/L. Spherical micelles of diameter 30–80 nm were obtained in aqueous solution, and prednisone acetate, an anti-inflammatory drug, was successfully loaded. Drug release from the micelles was sustained and continued to be up to 100 h. Heterogeneous reversible addition–fragmentation chain transfer (RAFT) [43] or ATRP [44] has been reported as a possible pathway to synthesize the cellulosegraft copolymer. However, grafting occurred only on the surface of cellulose fibers. Homogeneous ATRP has solved that problem. Graft copolymers from cellulose and its derivatives have been successfully synthesized. Cellulose-gPDMAEMA (polydimethylaminoethyl methacrylate) (Fig. 10.4) was synthesized using cellulose as macroinitiator for ATRP addition of DMAEMA [45]. Cellulose was acylated with 2-bromopropionyl bromide in a room temperature ionic liquid, 1-allyl-3-methylimidazolium chloride and acted as macroinitiator with CuBr/pentamethyldiethylenetriamine (PMDETA) as catalyst. The graft copolymer kept the pH and temperature responsiveness properties of PDMAEMA. Spherical aggregates with diameters in the range of 40–80 nm were observed in both transmission electron microscopy (TEM) and atomic force microscopy (AFM) images for the samples prepared from aqueous solution with concentration of 2 mg/mL at pH 7. Cellulose-g-poly(N,N-dimethylacrylamide) (PDMA) has also been synthesized using homogeneous ATRP [46]. PDMA has been chosen due to its biocompatibility and water solubility. Nanoparticles of size 200 nm were formed in aqueous medium. Similarly, derivatives of cellulose have been utilized in ATRP. Hydroxypropyl cellulose formed comb polymers via ATRP of methylmethacrylate. Globules of

364

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

N

O O

Br O O

O

m

O O

n

O

HO

n

O

O O

O

Br

HO

H2C

O

O

O

O

m O

N

O

N

FIGURE 10.4 Cellulose-g-PDMAEMA.

height 5 nm were obtained and this morphology was attributed to high grafting density [47]. Self-assembled amphiphilic ethyl cellulose-g-polyacrylic acid (EC-g-PAA) (Fig. 10.5) has been obtained again via ATRP [48]. EC-g-PAA copolymers can be self-assembled to micelles or particles with the diameter of 5 and 100 nm, respectively, in water (pH = 10, concentration = 1.0 mg/mL). pH-sensitive micelles are usually made from double-hydrophilic block copolymers. They can dissolve in water over a pH range and aggregate spontaneously upon an appropriate change in the pH value. Hydroxyethyl cellulose (HEC) grafted with polyacrylic acid gives rise to such a system [49]. The graft copolymer was synthesized using cerium(IV)-initiated free radical graft polymerization of acrylic acid from HEC. Perfect spherical micelles of size 53.8 and 75.6 nm were obtained depending upon the length of PAA at pH < 3. When pH was increased, a morphological transition to hollow spheres was observed (Fig. 10.6).

OCH2CH3 O O

H3CH2CO

n

O

Br m

O HO

O

FIGURE 10.5 Ethyl cellulose-g-polyacrylic acid.

MODIFIED POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES

365

pH > 3

pH < 3

FIGURE 10.6 pH-Dependent morphological change from spherical micelle to hollow sphere.

m

10.4.1.2 Polysaccharide Grafts Using Protection–Deprotection Method Acetylation has been a pathway adopted by some research groups. The first report of acetylation of a polysaccharide with the aim of producing amphiphilic micelles was made by Garcia and Vidal [50]. They have reported acetylation of agarose using acetic anhydride, determined degree of acetylation using infrared (IR) and nuclear magnetic resonance (NMR), and showed an increase in the hydrophobicity of the agarose. They did not, however, report on any deprotection and grafting. They suggested the use of the acetylated agarose itself as micelle for physical loading of drugs. Later the use of oligoagarose for grafting of polycaprolactone was reported [30a,b]. The welldefined oligoagarose was partially acetylated using the same technique as Garcia and Vidal, however, the unprotected hydroxyl groups were used as macroinitiator for the ring-opening polymerization of ε-caprolactone in the presence of Sn(Oct)2 . The acetyl groups were then removed, liberating hydroxyl groups, which resulted into amphiphilic star shaped micelles in the size range of 10-20 nm (Fig. 10.7).

m

O O

O

OH

O

O

O

OH O

O

O

O O O

O OH

m

O

FIGURE 10.7 Agarose-g-PCL [30].

n

366

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

OH O OCOCH 3

OH

OH O

OH

OCOCCH3 O

O

OH O OH

O OCOCHC3

OH OH

OH

O OH OH O

O O OCOCH3

OCOCH3 OH

O O OH

n

FIGURE 10.8 Acetylated pullulan.

Self-assembling nanospheres of hydrophobized pullulans have been prepared via acetylation (Fig. 10.8). Morphological studies observed by TEM showed that selfassembly of hydrophobized pullulans resulted in nice spherical shapes with a size range of about 50–100 nm depending on degree of grafting. The size and shape of this system was considered interesting for injectable drug targeting. A drug loading study was performed using clonazepam (CNZ) as hydrophobic model drug. CNZ was released from nanospheres via pseudo-zero-order kinetics, and the increased drug loading contents led to slower release of the drug [51]. Pullulan acetate was further decorated with vitamin H. The latter is present in higher amount in cancerous tumors compared to normal tissue. Rapid proliferation of cancer cells may require vitamin H, and the cell surface receptors may be overexpressed on tumor cells. Thus in an attempt to improve the design of self-assembled nanoparticles for enhanced cancer-targeting activity and internalization into cancer cells, vitamin H was incorporated [52]. Adriamycin was used as drug and indeed a strong absorption to HepG2 cells was observed. In a similar manner, folic acid (FA) has also been conjugated to pullulan acetate in order to improve cancer-targeting activity [53]. Ohya et al. developed polylactide-g-amylose [22] (Fig. 10.9), -pullulan [54], and -dextran [23] using the trimethylsilyl (TMS) protection–deprotection method. The hydroxyl groups on the polysaccharides were partially protected using TMS-Cl followed by ring-opening polymerization of lactide with t-BuOK. Deprotection to free hydroxyl groups was effected using methanol. While poly-l-lactide films did not

O

O

O

m

O OH

OH O OH

O OH

O

O O O

n

m

FIGURE 10.9 PLA-g-amylose.

MODIFIED POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES

367

absorb water at all because of their high hydrophobicity, the graft copolymer films swelled immediately after immersion in phosphate-buffered sulfate (PBS). The top surface of the graft-PLAs was suggested to be covered with hydrophilic segments, giving rise to a wettable surface. Such a wettable surface led to the suppression of cell attachment and protein adsorption onto the film. The copolymers exhibited better hydrophilicity and cell affinity compared to pure PLA. Nouvel et al. reported on a similar grafting procedure of polylactide on dextran using the three-step synthesis [24]. The dextran molecule was first protected via the silylation step, and the remaining unprotected hydroxyl groups were used to ring-open lactide using a catalytic amount of Sn(Oct)2 . The final step involved deprotection of the silylated OH groups. Depending on the number of PLA repeat units, PLA-grafted dextrans were soluble either in water or in organic solvents. Liu et al. have reported on the use of microwave for the grafting of ε-caprolactone onto chitosan via the phthaloyl protection method [29]. The resulting chitosan-gpolycaprolactone copolymer had free hydrophilic amino groups and hydrophobic polycaprolactone chains, as confirmed by NMR.

10.4.1.3 Polysaccharide Grafts Using Carboxymethyl Groups Polysaccharides grafted with carboxymethyl groups have been studied in a view of improving their performance as biomedical materials. Two such systems are carboxymethyl chitin [55] and chitosan [56]. For instance, carboxymethyl chitin was found to be nontoxic and was successfully used to load the anticancer drug 5-fluorouacil [55]. The prepared nanoparticles were found to be antibacterial and their magnetic properties revealed their potential use in drug tracking. The nanoparticles showed a spherical morphology with a diameter of 200–250 nm. The drug encapsulation efficiency was 78.3% and the amount of drug loaded was 6 wt% of the total base polymer. The in vitro drug release studies were carried out at pH 6.8, which is similar to the pH of the surroundings of the tumor mass. The release mechanism from such a system was proposed to occur via diffusion and degradation of the polymer. O-carboxymethylated (CM) chitosan is a water-soluble polymer where the H of the hydroxyl group of the monomer is replaced by a carboxymethyl group through an ether bond. The biocompatibility of this material has already been proven [57]. It is prepared by chemical reaction of chitosan with monochloroacetic acid [58]. The water solubility of the CM chitosans is closely related to the reaction conditions and degree of carboxymethylation. The CM chitosans, prepared at temperatures of 0–10◦ C, are soluble in water while CM chitosans prepared between 20 and 60◦ C are insoluble in the water at near-neutral pH. The increase in reaction temperature increased the degree of substitution and thus decreased solubility at lower pHs. At higher pHs, insolubility resulted from a lower degree of substitution. Folic acid has been conjugated on carboxymethyl chitosan to facilitate internalization by receptor-mediated endocytosis as it is known that folic acid receptors are overexpressed in many types of human cancer cells (Fig. 10.10) [58]. Doxorubin was successfully loaded in the nanoparticle formed by the folic-acid-conjugated carboxymethyl chitosan (150 nm) [59]. Excellent stability in aqueous medium over a wide range of physiological conditions with reasonably good hydrodynamic size was

368

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

O

O

N H

NH2

O

O

H N

Folate

O OH

OH O

HO

O

O n NHCOCH3

FIGURE 10.10 Folic acid conjugated carboxymethyl cellulose.

obtained. Release behaviors of doxorubicin from nanoparticles showed pH dependence and a sustained release pattern. More effective targeting of cancerous cells was detected. The effect of degree of substitution of carboxymethyl chitosan and molecular weight (MW) on doxorubicin delivery have also been investigated [60]. Spherical particle of sizes of 200–300 nm were obtained and loading efficiency increased from 10 to 40% with an increase in MW from 4.5 to 38.9 kDa. Degree of substitution had a slight effect on loading. However, the release rate was hampered by the high molecular weight and degree of substitution.

10.4.1.4 End-to-End Coupling of Polysaccharides with Preformed Polymers/Small Chains End-to-end coupling in the presence or absence of coupling agents to form graft polysaccharides for drug loading devices has been reported in order to produce amphiphilic materials. Biodegradable amphiphilic copolymers based on polycaprolactone (PCL) and chitosan were synthesized and were used to successfully prepare nanoparticles [27]. The PCL-g-chitosan copolymers were synthesized by coupling the hydroxyl end groups on preformed PCL chains and the amino groups present on 6-O-triphenylmethyl chitosan and by removing the protective 6-O-triphenylmethyl groups in acidic aqueous solution. The PCL content in the copolymers could be controlled in the range of 10–90 wt%. The copolymers were shown to form spherical or elliptic nanoparticles in water. Another type of core–shell micelle forming graft copolymer based on polycaprolactone as polyester and chondroitin sulfate (CS) as polysaccharide has been synthesized using the end-to-end coupling method [61]. Chondroitin sulfate is a highly hydrophilic polymer that is biocompatible and anti-inflammatory. The rationale behind the addition of PCL to CS was to increase segregation of the two segments, which would result into low critical micelle concentration (CMC) values and thus increase stabilization of nanoparticles in physiological medium. The coupling of the double-bond end of the preformed PCL with a vinyl group on CS via a radical reaction was used (Fig. 10.11). The particle sizes of micelles were in the range of 170–300 nm depending on the number of PCL chains. They neither aggregate nor change in hydrodynamic sizes after 15 days in solutions containing salts or polyvinyl alcohol (PVA). Cytotoxicity as determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay demonstrates that KB cells (oral epidermoid cell) incubated with the PCL-CS micelles for 24 h remained approximately 100% cell viable at a concentration of 10 ␮g/mL. Bovine serum albumin (BSA) was loaded

MODIFIED POLYSACCHARIDES AS NANODRUG DELIVERY DEVICES

369

COOH O OH

COOH O OH

O

O

OSO3 Na+ OH

O

O O

n

NHCOCH3

O n

NHCOCH3

O

OH

O

O

O

OSO3 Na+

O O

O O O O m

O O m

FIGURE 10.11 CS-g-PCL.

as model protein drug and a higher encapsulation and loading efficiency than PCL-gdextran [62] was obtained, showing that better segregation between hydrophilic and hydrophobic segments lead to higher drug loading capacity. PCL-g-DEX has been reported using end-to-end coupling of polymers (Fig. 10.12) in view of assessing a polysaccharide-coated particle for protein interaction [63a,b]. Monocarboxylic acid end-capped PCL was reacted with carbonyl diimidazole. The resulting imidazolide was then reacted with dextran (DEX) at different molar ratios to obtain amphiphilic copolymers with various hydrophilic–lipophilic ratios. The hydrodynamic diameter of the PCL-g-DEX5000 and PCL-g-DEX40000 were 196 and 260 nm, attributed to the thickness of the DEX shell and its hydration behavior. BSA was loaded as a model protein and the protein adsorption was two and four times lower than uncoated PCL depending on copolymer composition. Shorter chains have also been grafted on oligosaccharides using end-to-end coupling. One such example is chitosan oligosaccharide-g-stearic acid [64]. The carboxyl group on stearic acid was condensed on an amino group of oligochitosan using

O

O O

HO HO O PCL

O

HO HO n

OH

O O

N

PCL N

FIGURE 10.12 PCL-g-DEX.

n

370

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

FIGURE 10.13 Crosslinked shell-oligochitosan-g-stearic acid.

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the coupling agent. The graft copolymers formed spherical micelles that decreased in size from 74.6 to 28.1 nm with an increase in degree of substitution (5, 12, and 42%). The shell of the micelles (chitosan) was further crosslinked with glutaraldehyde (Fig. 10.13). An increase in particle size after crosslinking was only observed at high concentration of glutaraldehyde (110 nm). Paclitaxel was loaded in the hydrophobic core of the micelles. An increase in particle size was observed (up to 126.5 nm) and high encapsulation efficiency ranging from 94.82 to 99.14% was obtained. The release of Paclitaxel was studied in both the crosslinked and non-crosslinked micelles. The non-crosslinked micelle showed an inversely proportional relationship between degree of substitution and release time (DS = 42%, t = 24 h, DS = 5%, t = 8 h). The high degree of substitution caused an increase in hydrophobic interaction between the hydrophobic drug and the hydrophobic segment of micelle (stearic acid), resulting in a decrease in release rate. Crosslinking the shell brought about a further decrease of the release rate.

10.5 MODIFIED POLYSACCHARIDES—BLOCK SYSTEMS A polymeric micelle is a macromolecular assembly composed of an inner core and an outer shell and most typically is formed from block copolymers. In the last two decades, polymeric micelles have been actively studied as a new type of drug carrier system, in particular for drug targeting of anticancer drugs to solid tumors. Fewer block polysaccharide systems have been reported compared to graft systems. There are three possible strategies to achieve block copolymerization. One uses coupling of prefabricated blocks where the polysaccharides have to be end-capped with a specific group before it can form block copolymers using the end-to-end coupling technique in a good solvent for both blocks. A second strategy is based on protection–deprotection of the OH groups before/after block copolymerization can occur. Another strategy consists of end-capping the protected polysaccharide and using it as macroinitiator for polymerization of the second monomer, resulting in the block copolymer. The first report of a block copolymer was made by Ziegast and Pfannem¨uller [65]. They synthesized a PEO-b-oligosaccharide using end-to-end coupling via condensation of

MODIFIED POLYSACCHARIDES—BLOCK SYSTEMS

371

OH

OH

OH

O

OH O

O

OH

O

NH

n OH

O

m

OH

FIGURE 10.14 PEO-b-oligosaccharide.

a saccharide unit to lactone and its subsequent binding to an amino end-capped PEO by amide linkage (Fig. 10.14). Out of the reported block systems only a very few have undergone self-assembly in water to give micellar [66–70] or vesicular [71, 72] aggregates, and one system showed self-assembly in chloroform [73].

10.5.1 Oligosaccharide-b -Copolymer Maltoheptaose-b-poly(ε-caprolactone) (MH-b-PCL) copolymer (Fig. 10.15) has been synthesized using peracetylated maltoheptaose [74]. The protection–deprotection approach was adopted. All hydroxyl groups on maltoheptaose were acetylated before selective deprotection of the hydroxyl group at the reducing end. The latter was used as macroinitiator to initiate the ring-opening polymerization of ε-caprolactone in the presence of tin(II) octanoate as catalyst. Deprotection of the remaining hydroxyl groups after copolymerization yielded an amphiphilic copolymer. Spherical micelles of size 117.6–123.7 nm were obtained depending on the length of the PCL block. Vesicle morphology was reported for maltoheptaose-b-poly(N-isopropylacrylamide) (Fig. 10.16, Mal7 -b-PNIPAMn) copolymers with a diameter of 300 nm [72]. The self-assembly of this system is favored due to the hydrophobicity of PNIPAMn above the cloud point (36.4–51.5◦ C). The end-to-end technique based on click chemistry was used to synthesize the block copolymer. Alkyne-functionalized maltoheptaose and azide-functionalized PNIPAMs were reacted together using sodium ascorbate and copper sulfate as catalysts at room temperature.

OH HO HO

O OH OH

O

O OH

HO OH

O 5 HO

O O OH

O

FIGURE 10.15 Maltoheptaose-b-PCL.

O O

O n

372

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

OH HO

HN

OH

HO

OH

O

O H N

Ac N

6 HO

N3

n Cl

O

OH O

Cl n

HN O N

NH

N

OH N

HO OH

HO

OH

O

AcN

6 HO

OH

FIGURE 10.16 Mal7 -b-PNIPAMn .

10.5.2 Dextran-b -Copolymer The most common polysaccharide used for the purpose of block copolymerization is commercial dextran due to its terminal aldehyde, which can be modified using reductive amination or oxidation. This aldehyde moiety can also serve as anchoring group to assemble a diblock copolymer. The synthesis of dextran-b-polystyrene was first reported using the coupling of dextran and amino-terminated polystyrene (Fig. 10.17) [75]. The reductive amination technique was chosen to couple the two blocks, and sodium cyanoborohydride was the reducing agent. However, this technique prevented coupling of dextran chains with Mw > 6000. The intrafacial pressure recorded for the copolymer was different from the individual polymers and pointed toward the formation of an amphiphile. O

n

O

HO HO

OH HO HO

O OH OH

H N

Si m

FIGURE 10.17 Dextran-b-polystyrene using prefabricated blocks.

MODIFIED POLYSACCHARIDES—BLOCK SYSTEMS

373

One method of protection using silylation/desilylation of dextran for its block copolymerization with styrene has been reported by Houga and co-workers [67, 68]. They generated a bromoisobutyramide-ended dextran from a commercially available sample using the terminal anomeric aldehyde. Before growing the PS block by ATRP, the OH groups of the dextran were silylated to make it soluble in regular organic solvents. Next, styrene was polymerized from the corresponding silylated dextran-based ATRP macroinitiator using CuBr/PMDETA as catalyst. The block copolymer (Fig. 10.18) adopted a micelle-like spherical shape with a diameter of 56 nm. However, as the polystyrene content was increased, the morphology changed from core–shell micelles to ovoids to vesicles. The PS weight fraction ranged from 7 to 92% w/w, whereas the average number molar mass of dextran was kept constant at 6600 g mol−1 . Self-assembly by direct dissolution in water could be performed only for block copolymers with a low hydrophobic content (7% w/w), whereas mixtures of tetrahydrofuran and dimethylsulfoxide were required for higher PS content, before transferring the structures into water. Schatz et al. synthesized a dextran-b-PBLG (poly(y-benzyl l-glutamate)) using an end-to-end technique based on click chemistry. The copolymer (Fig. 10.19) selfassembled in water into small polymersomes with a membrane thickness of 23 nm [71]. The strategy for block polymerization was based on adding together through click chemistry an azido end-capped PBLG and an alkyne end-capped dextran. The alkyne group was introduced at the reducing end of the dextran (Mn = 6600 g mol−1 ). The PBLG end-functionalized with an azide group had a degree of polymerization DP = 59 and was obtained through the ring-opening polymerization of ␥ -benzyl-lglutamate-N-carboxylic anhydride.

OH HO O HO

HO HO O HO

HO

n OH NH

HO HO

HO

O NH

Br m

FIGURE 10.18 Dextran-b-polystyrene and corresponding TEM (reproduced by permission of The Royal Society of Chemistry) from water solution.

374

HO

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

m

O OH OH

O

OH

OH

OH NH

OH OH

O N

N N

H N H n

N H O O

FIGURE 10.19 Dextran-b-PBLG.

Upadhyay et al. adopted the same strategy using a different polysaccharide to yield a drug-loaded polysaccharide-containing block segments [76]. They synthesized poly(␥ -benzyl-l-glutamate)-b-hyaluronan (PBLG23 -b-HYA10 ) using a similar click chemistry technique as for their dextran-b-PBLG. Polymersomes were obtained by self-assembly of the copolymers, forming bilayer membranes, where the hydrophilic hyaluronan blocks are exposed to the water interface. Doxorubicin was successfully loaded in the polymersomes (12 wt%) with an encapsulation efficiency of 50% (Fig. 10.20). The hydrodynamic volume of the Dox-loaded polymersome was 220 nm. The intracellular delivery of Dox was studied using high (MCF-7) and low (U87) CD44 expressing cancer cell models. The copolymer without Dox was nontoxic at concentrations of 150–650 ␮g/mL. The internalization mechanism of the polymersomes with Dox (PolyDox) and free Dox was followed by fluorescence microscopy. Release of Dox from the polymersome was mostly by diffusion through the bilayer membrane. The images recorded showed that PolyDox and free Dox have different internalization mechanisms. PolyDox was taken up by cells via an endocytic pathway and was localized in acidic endocytic compartments. Dox showed rapid intercalation to the chromosomal deoxyribonucleic acid (DNA) after passive diffusion into the cells. A decrease in Dox concentration was observed in the nuclei and cytoplasm of U87 cells while a high level of PolyDox was present in the cytoplasm of MCF-7 cells. The authors have concluded that free Dox was expelled by the p-glycoprotein pump present in tumor cells U87, whereas the copolymer most probably reversed the multidrug resistance induced by p-glycoprotein. Liu and Zhang have reported on the synthesis of a novel dextran-bpolycaprolactone (Fig. 10.21), which can self-assemble into spherical micelles of size 20–50 nm [66]. They made use of an acrylol end-capped PCL chain and an aminofunctionalized dextran, which they reacted together in a Micheal addition reaction.

MODIFIED POLYSACCHARIDES—BLOCK SYSTEMS

O

375

O

OH

OH OH

OCH3

O

OH

OH

PBLG

O

HYA

OH NH2

FIGURE 10.20 PolyDox.

Sun et al. synthesized dextran-b-PCL using a disulfide bridge to link the two blocks yielding a DEX-SS-PCL (Fig. 10.22) [70]. The aldehyde end of dextran was converted to orthopyridyl disulfide (DEX-SS-Py), and it was reacted with mercapto PCL (PCL-SH). Micelles of the copolymer prepared by the solvent exchange method had a size of 60 nm and the CMC was 9.3 mg/L. The anticancer drug doxorubicin was loaded into the micelles by the dialysis method with a loading efficiency of 70% and an increase of micelle size to 80 nm. The cellular uptake and intracellular release behaviors of DOX-loaded DEX-SS-PCL micelles were followed with fluorescence microscopy and confocal laser scanning microscopy using a mouse leukemic monocyte macrophage cell line (RAW 264.7). Fast internalization and rapid release of Dox was noted (after 2 h). These results were compared with Dox-loaded DEX-b-PCL, which showed a low concentration of Dox after 4 h. The difference between the two copolymers was attributed to the presence of the disulfide bridge, which is cleaved in the intracellular compartments (cytosol) and the cell nucleus due to the presence O HO HO

O OH HO HO

O n OH N OH H

H N

O O

FIGURE 10.21 Dextran-b-PCL.

O

m OCH2CH2CH3

376

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

O

O

HO HO

O n

OH

OH

HO HO

N OH H

O S

O

S

O O

m

(CH2)5OH

FIGURE 10.22 DEX-SS-PCL.

of comparatively high concentrations of reducing glutathione tripeptides. Dox is released from the micelle upon cleavage of the dextran portion. It has to be noted that free Dox mainly accumulates in the cell nucleus. MTT assays using HepG2 and RAW 264.7 cells revealed that Dex-SS-PCL micelles were practically nontoxic up to a tested concentration of 3.0 mg/mL. Double hydrophilic block copolymers (DHBC) were prepared by end-to-end coupling of two biocompatible water-soluble homopolymers of different chemical nature: polysaccharide carboxymethyl dextran (Mw = 8300) and ␻-amino poly(ethylene glycol) (Mw = 3000) [69]. Carboxymethyl dextran was linked to poly(ethylene glycol) and the two blocks were able to undergo noncovalent interactions via H bonding. The synthetic strategy involved, first, amidation with ␻-amino methoxy poly(ethylene glycol) of end-lactonized dextran (DEXelactone) leading to neutral dextranepoly(ethylene glycol)s (DEXePEG) and, second, carboxymethylation of the dextran block to produce carboxymethyldextranepoly(ethylene glycol) (Fig. 10.23). This copolymer offers the advantage of being pH responsive due to the presence of COOH moiety. This characteristic is extremely useful for drug delivery devices used for oral route due to the acidic pH of the gastrointestinal tract. In acidic or neutral

x

O HO

O

HO

OH

pH < 5

Intra- and interchain H bonding between COOH &

5 < pH < 9

Intrachain H bonding between COOH &

pH > 9

Interchain H bonding between

OH glucose and O

OH glucose and

OH glucose and

O PEG

O O

HO O COOH

OH HO HO

OH

O

O COOH y n

OH

OH

O

OH

N H

O m

FIGURE 10.23 pH responsive CMD-b-PEG copolymer.

O

PEG

PEG

MODIFIED POLYSACCHARIDES—BLOCK SYSTEMS

O

OH OH

CH2OH O OH

CH2OH O OH

CH2OH O OH

OH

O * n

O NH

O

377

O 110

OH

FIGURE 10.24 MPEO-b-amylose.

conditions this system exists in the form of micelles, while in alkaline conditions the assemblies contained at most three polymer chains. 10.5.3 Amylose-b -Copolymer The first synthesis of a block ␻-methoxypoly(ethylene oxide)-amylose copolymer (MPEO-amylose) (Fig. 10.24) was reported by Akiyoshi et al. [77]. This copolymer is interesting as it contains an amylose end group that can act as a recognition site. The copolymer was synthesized by an enzymatic reaction using potato phosphorylase from an MPEO-maltopentaosylamine derivative as a primer and ␣-d-glucose1-phosphate as a substrate. Contrary to amylose, which usually aggregates in water and precipitates due to the formation of an intermolecular double helix, these MPEOamyloses did not aggregate even after 1 day. They formed complexes with iodine in water, and an MPEO-amylose-iodine complex solution was stable for at least a month without precipitation. The authors suggested micellar formation in water. Later the solution properties of the amphiphilic methoxy poly(ethylene oxide)-blockamylose were studied [73]. They showed self-assembly in chloroform (19–28.8 nm) and methyl orange was successfully encapsulated. Loos and Stadler were the first to report on the synthesis of an amyloseb-polystyrene copolymer [78]. They reacted amino-functionalized polystyrene with maltoheptaonolactone followed by enzymatic polymerization using potato phosphorylase to extend the maltoheptaose primer (Fig. 10.25a). Later another pathway was reported for the synthesis of the same copolymer using a silane endfunctionalized polystyrene (Fig. 10.25b) and the following as catalysts: bis(1,5cyclooctadien)dirhodium(I) dichloride, bis(dibenzylsulfido)platinum(II) chloride, octacarbonyl dicobalt, and Speier’s catalyst [79]. The successful synthesis of amylose60 -b-polystyrene740 (Fig. 10.25a) with a higher ratio of polystyrene than amylose has been reported by Loos et al. [80] using the technique of Loos and Stadler [78]. Since the hydrophobic portion is longer than the hydrophilic part, crew-cut micelles [81–83] (Fig. 10.25a) of diameter 10–30 nm were obtained in water. These crew-cut micelles have a large hydrophobic core and a thin hydrophilic corona. The structures of these micelles differ from classical ones by the rodlike, helical structure of the corona. 10.5.4 Hyaluronan-b -Copolymer Hyaluronan-b-poly(2-ethyl-2-oxazoline) (Fig. 10.26) has been synthesized as a novel biomimetic diblock copolymer consisting of two hydrophilic units: a neutral and

378

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

OH O

HO HO

OH OH O

O

HO

OH

OH

OH

O

n HO

Hydrophilic corona

HO

H N

(CH2)3 m

O

Hydrophobic core

Crew-cut micelle

(a) OH O

HO HO

OH OH O

O

HO

OH

OH

OH

O

H N

n HO

HO

Si (CH3)3

m

O

(b)

FIGURE 10.25 Amylose-b-polystyrene.

COOH OH

O

OH

CH2OH O

O

OH

O

O

OH CH2NH

CH2 CH2

OH

OH OH

CH2OH

COOH

O

NHAc

NHAc

OH

N CH2 CH2 O

n

C

N

CH2 CH3

CH3

HA-b-PEtOz N

N

H N

H2N

NH2 NH

CH2

mC O CH2

NH

Diminazene

FIGURE 10.26 HA-b-PEtOz copolymer and diminazene.

CH2 OH

MODE OF DELIVERY OF MODIFIED POLYSACCHARIDE NANOPARTICLES

379

an anionic block [84]. Poly(2-alkyl-2-oxazolines) are nontoxic neutral hydrophilic polymers with excellent biocompatibility and have found applications as components of drug delivery systems. Coupling of the two preformed polymers was used as a technique of block copolymerization. Reductive amination of the hyaluronan (HA) terminal aldehyde group with a primary amine linked to poly(2-ethyl-2-oxazoline) (PEtOz) was applied. The ratio of the HA to PEtOz blocks in the resulting copolymer was determined to be 1:10.5. The copolymer formed stable colloidal particles of size 130 nm with the cationic drug diminazene.

10.6 MODE OF DELIVERY OF MODIFIED POLYSACCHARIDE NANOPARTICLES The drug administration pathways using polymers/copolymers are via oral, inhalation, transdermal, implantation, and injection routes. In this section, we will discuss the potential use of modified polysaccharides as oral and nasal drug delivery systems, which are more patient friendly than the other techniques. 10.6.1 Oral Drug Administration The oral delivery of drugs is regarded as the optimal means for achieving therapeutic effects owing to increased patient compliance. After oral administration, both the drug and the carrier system have to overcome many barriers before reaching the systemic circulation such as gastrointestinal (GI) destruction of labile molecule and low levels of macromolecular absorption. To reduce the impact of digestive enzymes and to ensure the absorption of bioactive agents in an unaltered form, molecules may be incorporated into polymeric drug carriers. Polymeric micelles represent a promising delivery vehicle especially intended for poorly water-soluble pharmaceutical active ingredients in order to improve their oral bioavailability. They have been used to protect drugs during transport through the acidic environment of the stomach and improve transport across the intestinal wall. Drug delivery across the intestinal barrier occurs via three pathways: paracellular passive diffusion, transcellular passive diffusion, and transcellular receptor-mediated transcytosis [85]. Internalization depends on size, chemical composition, and charge of drug carriers. Prior to transcytosis and translocation a number of other factors affect nanoparticle uptake: transit times in the GI tract, residence times in regions of particle uptake, transport through mucus, and stimulus for cellular uptake [86]. These factors affect absorption and delivery of nanoparticles. Typically, requirements for nanoparticles are size less than 500 nm, reasonable hydrophobic segment length, and presence of charged moiety. Particle size is an important consideration when designing a polymeric carrier; below a certain size there is a lack of optimization of interaction of surface receptors and epithelial lining and above a certain size range diffusion through cell matrix becomes problematic. Three polysaccharides have been mostly proposed for this purpose: modified chitosan, modified dextran, and hydropropylcellulose. For best results these naturally

380

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

available and nontoxic polymers have been chosen. Chitosan is regarded as safe and biocompatible. Moreover, due to its cohesive properties it can be used in matrix tablets in order to provide controlled release. Chitosan is capable of opening tight junctions and can therefore improve the oral uptake of hydrophilic drugs such as peptides. It is also widely used due to its mucoadhesive properties. Dextran is inert in biological systems and does not affect cell viability. Biodegradability at specific body sites, for example, the colon makes dextran an ideal candidate for oral drug delivery systems. However, due to its water solubility it has to be rendered hydrophobic before it can be used as a drug carrier. Hydroxypropyl cellulose is widely used as an excipient in oral solid dosage forms in which it acts as a disintegrant and as a binder in granulation. It is essentially a nontoxic and nonirritant polysaccharide [87].

10.6.1.1 Modified Chitosan One area where oral administration is receiving major attention is insulin delivery. At present, it requires single or multiple daily subcutaneous injections to achieve the desired therapeutic effect, which is inconvenient and painful and with poor patient compliance. The therapeutic efficiency of insulin can be increased by encapsulation in a sustained dosage form that is capable of releasing the drug continuously and at a controlled rate. To this effect insulin encapsulation and release have been studied using hydrophilic nanoparticles consisting of chitosan and the monomers methyl methacrylate (MMA), N-trimethylaminoethyl methacrylate chloride (TMAEMC), and N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC) (Fig. 10.27) [88]. The resulting nanoparticles were of size 150–280 nm and carried positive surface charges. Encapsulation efficiency was up to 100%. In vitro release showed an initial burst followed by slow sustained release for more than 24 h. The graft copolymer nanoparticles enhanced the absorption and improved the bioavailability of insulin via the GI tract of normal male Sprague–Dawley rats to a greater extent than that of the phosphate buffer solution (PBS) of insulin. Another device consisting of insulin-loaded dextran sulfate/chitosan nanoparticles has been evaluated following oral dosage in diabetic rats [89, 90]. The nanoparticles were mucoadhesive and negatively charged with a mean size of 500 nm, suitable for uptake within the gastrointestinal tract. The mucoadhesive and absorption enhancement properties of chitosan and the protective effect of dextran sulfate against

H3C RO O

OH

HN

O

n

HO

O

O

HO HN H3C RO O

OH

O

R = -CH2CH2N(CH3)3+Cl-

m

+

TMAEMC -

R = -CH2CH2NH(CH3)2 Cl

n

FIGURE 10.27 Chitosan-g-TMAEMC and DMAEMC.

DMAEMC

MODE OF DELIVERY OF MODIFIED POLYSACCHARIDE NANOPARTICLES

381

insulin release at low pH were combined to promote and ensure insulin intestinal absorption. Adhesion to rat intestinal epithelium and internalization of insulin within the intestinal mucosa was shown using confocal microscopy. These nanoparticles lowered serum glucose levels of streptozotocin-induced diabetic rats at insulin doses of 50 and 100 IU/kg up to 67 and 64%, respectively, of their basal glucose level. The hypoglycemic effect and insulinemia levels were higher by two- to threefold than those obtained from oral insulin solution. In addition, the hypoglycemic effect was observed for more than 24 h. An extensive review of modified chitosans for oral drug delivery has been published by Werle et al. [91]. Another interesting review has been made by Prabaharan and Mano on the potential of chitosan-based particles as a controlled drug delivery system [92]. Chitosan has been modified to either improve its features, for example, solubility at different pH or add new functionalities. The free amino group has been used to perform chemical additions on chitosan. For instance, N-trimethyl chitosan chloride (TMC) (Fig. 10.28) has been synthesized to improve the solubility over a broader range of pH [93]. Indeed, the presence of the trimethyl chloride made chitosan soluble even at basic pH, which is valuable for oral drug administration [94]. Absence of toxicity and decrease in transepithelial resistance (opening of the tight junctions) of Caco-2 cells were observed. Caco-2 cells are generally selected to estimate in vivo drug absorption, as they retain many features of small intestinal epithelial cells. However, this was accompanied by a decrease in mucoadhesiveness. Insulin has been loaded using TMC yielding particles with size 100–320 nm [95]. It was found that insulin was protected against enzyme digestion in the presence of trypsin using TMC. To improve the mucoadhesiveness of chitosan, thiolated derivatives were synthesized (Fig. 10.29) [96]. Iminothiolane-chitosan was shown to be in vitro approximately 130-fold more mucoadhesive than unmodified chitosan. The rationale behind this synthesis was the possible formation of a disulfide bridge between free thiols on chitosan and the mucus lining [97]. The toxicity profile was similar to unmodified chitosan. Insulin has also been successfully conjugated on thiolated chitosan (Chitosan–TBA (4-thiobutylamidine)) and Chitosan–TBA–insulin tablets fed to nondiabetic rats [98]. It significantly decreased the blood glucose level of nondiabetic rats for 24 h corresponding to a pharmacological efficacy of 1.69 ± 0.42% (means ± SD; n = 6) versus subcutaneous injection. Controlled release of insulin was observed over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohesive properties of chitosan were at least 60-fold improved by the immobilization of thiol groups on the polymer. OH O

HO

H2 N O

O

HO N

Cl

OH

O

m

FIGURE 10.28 N-trimethyl chitosan chloride.

382

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

OH O

H2N

HO

O

O

HO

O

OH

HN

m

NH

HS

FIGURE 10.29 Thiolated chitosan.

10.6.1.2 Modified Dextran Dextran can be degraded by the enzyme dextranase in the colon. This has resulted in the design of polymeric prodrugs or nanoparticles for colonic drug delivery based on dextran. Poly(dl-lactide-co-glycolide) (PLGA) grafted dextran (Fig. 10.30) formed uniform spherical core–shell nanoparticles in water by self-assembly with particle size 245.3 nm [99]. Size and morphology provided them with acceptable properties for use as drug-targeting carriers. The core–shell nanoparticles were loaded with clonazepam. The drug-loaded particles showed an increase in size to 427.5±127.8 nm. Drug release from the core–shell type of nanoparticles was faster in the presence of dextranase, indicating that core–shell type of nanoparticles of PLGA-g-dextran can be used as oral drug carriers. Variation of the ratio of grafting resulted in spherical core–shell micelles of size varying from 50 to 300 nm. The anticancer drug doxorubicin was loaded in these polymeric micelle [100]. The drug release rate from the nanoparticles was again faster in the presence of dextranase.

O OH O

O OH

O O OH

O OH O

n O

O O

O O H m

x

O n O

O H m

FIGURE 10.30 Poly(dl-lactide-co-glycolide) (PLGA)-grafted dextran.

383

MODE OF DELIVERY OF MODIFIED POLYSACCHARIDE NANOPARTICLES

O OH

O

OH

n

O O 10

C15H31

FIGURE 10.31 Dextran-g-PEO10 -C16 .

Dextran-g-PEO10 -C16 has been designed using PEO as linker to attach the hydrophobic cetyl groups resulting in an amphiphilic material (Fig. 10.31) [85, 101]. The size of the micelles formed varied between 10 and 45 nm depending on the length of dextran used. These micelles were evaluated for the oral administration of poorly soluble cyclosporine A, a highly effective immunosuppressive agent used after organ transplantation to avoid graft rejection. Oral administration of cyclosporine A is affected by metabolizing enzymes: cytochrome P-450 3A4 (CYP3A4), the multidrug transporter P-glycoprotein (PGP) in the small intestine, and hepatic CYP3A4 thereby limiting absorption through the GI mucosa [102]. The clinical importance of cyclosporine A has driven research to obtain oral formulations leading to acceptable bioavailability. The amount of drug encapsulated increased with the number of octadecyl-PEO moieties grafted on dextran. Since oral administration was targeted, Caco-2 cells were used for cytotoxicity study. DEX-g-PEO10 -C16 exhibited no significant toxicity toward Caco-2 cells, up to concentrations of 10 g/L. Vitamin B12 was then linked to the DEX-g-PEO10 -C16 using 2,2 (ethylenedioxy)bis(ethylamine) as spacer (Fig. 10.32) [33]. The rationale for attaching vitamin B12 was to use its endogenous absorption pathway to enhance absorption of cyclosporine A. Indeed, due its size vitamin B12 is transported through the intestine through receptor-enhanced endocytosis, which is initiated by its complexation to intrinsic factor (IF), a protein produced in the stomach. In the small intestine this

O HO O O

C15H31

OH

10

n O O

HO O

O Vitamin B12

O

HN

O

O

H N

O O

FIGURE 10.32 VitB12 -modified-DEX-g-PEO10 -C16 .

OH

m

384

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

complex will then bind to IF receptors stimulating its internalization. Thus vitamin B12 (VB12 ) was covalently added to the corona of the DEX-g-PEO10 -C16 micelles and cyclosporine A (CsA) was present in the core. The permeation of CsA loaded within VB12 micelles (size = 33 nm) through intestinal enterocytes was evaluated in vitro using the human colon adenocarcinoma, Caco-2, cells in the presence of IF. Following 24 h of transport, the amount of transcytosed CsA is twice as large in the case of VB12 -modified micelles, compared to the naked micelles.

y

10.6.1.3 Modified Cellulose Cellulose derivatives are another class of suitable material for the oral delivery of drugs. Hydroxypropyl cellulose-g-polyoxyethylene alkyl ether [HPC-g-(POE)y -Cn ] (y = 10 or 20, n = 16 or 18) polymeric micelles have been used to encapsulate poorly water-soluble drugs in order to improve their oral bioavailability (Fig. 10.33) [85, 103]. The size of CsA-loaded micelles varied with degree of grafting and length of y and n (50–75 nm). The loading efficiency also varied due to the same parameters (15.5–21%). No toxicity was observed up to a concentration of 10 g/L using Caco-2 cells. The absence of toxicity toward intestinal cells represent promising characteristics for the development of a novel polymeric drug carrier for the oral delivery of poorly water-soluble drugs. The effect of viscosity of HPC on oral bioavailability of insulin has been studied [104]. Viscosity was varied by using HPC of different degrees of substitutions [GF (low), MF (medium), and HF (high)]. It was found that the rate and extent of oral insulin absorption from systems with HPC were dependent on the viscosity grade. Significant reduction of blood glucose level in rabbits was recorded using the mediumviscosity grade (MF) preparation. The release of insulin is diffusion controlled; thus

H2n+1Cn

O

O

O

O O OH

O

O

O

O

OH

O

O O

CnH2n+1 y

m

O HO O

O

O

CnH2n+1 y

FIGURE 10.33 [HPC-g-(POE)y -Cn ] (y = 10 or 20, n = 16 or 18).

MODE OF DELIVERY OF MODIFIED POLYSACCHARIDE NANOPARTICLES

385

the high-viscosity HPC resulted in slower diffusion rate and slow clearance from absorption site. The low-viscosity HPC gave less absorption-enhancing effect for insulin, due to faster clearance and the lower concentration gradient at the site of absorption. 10.6.2 Nasal Administration Oral administration offers a convenient pathway for the administration of a variety of drugs ensuring high patient compliance. However, drugs such as peptides and proteins are degraded by the proteolytic enzymes in the stomach, and most of these drugs need to be administered repeatedly by injection, for example, insulin. Intranasal drug delivery offers an alternative convenient method that has many advantages, such as a large absorptive surface area and high vascularity of the nasal mucosa, where drugs absorbed from the nasal cavity pass directly into the systemic circulation, thereby avoiding first-pass liver metabolism and quicker onset of pharmacological activity [105, 106]. There are many local diseases of the lung that are prime candidates for inhalation therapy, such as asthma, chronic obstructive pulmonary disease (COPD), primary pulmonary hypertension, and cancer. Asthma is a chronic inflammatory disease of the airway characterized by the infiltration of eosinophils, epithelial hyperplasia leading to hypersecretion of mucus, and the presence of airway hyperresponsiveness (AHR) to a variety of stimuli. Illum et al. [107] have used chitosan to enhance nasal absorption of polypeptides such as insulin in rats and sheep models. The adhesion properties of chitosan and transient widening of the tight junction were suggested to have played a major role in absorption. Chitosan has also been shown not to cause any membrane or cellular damage in a rat perfusion model [108]. Transient inhibitory effect on mucocilliary transport rates has also been reported using chitosan and chitosan glutamate solutions applied to human nasal tissue both ex vivo and in vivo [109]. Thiolated chitosan nanoparticles (220 ± 23 nm) have been assessed for the treatment of asthma using a mouse model of allergic asthma [110]. As discussed earlier, the presence of thiol groups on chitosan increases its mucoadhesiveness and permeation properties without affecting biodegradability [98, 111, 112]. Theophylline, a widely prescribed antiasthmatic drug, has been loaded into the thiolated nanoparticle in an attempt to increase its absorption by the bronchial epithelium and also its residence time. The loaded nanoparticle was administered via an intranasal pathway to ovalbumin (OVA)-challenged mice to produce an inflammatory allergic condition. It was observed that the anti-inflammatory effects of theophylline were markedly enhanced when the drug was delivered by thiolated chitosan nanoparticle compared to unmodified chitosan or theophylline alone (Fig. 10.34). Inhibition of infiltration of inflammatory cells beneath the epithelium and reduced epithelial damage were observed in ovalbumin-allergic mice, which were attributed to increased mucoadhesiveness. Another derivative of chitosan, hexanoyl chitosan, was synthesized through a coupling reaction between chitosan and hexanoic anhydride in order to investigate its use as protein carriers for nasal drug delivery [113]. Hexanoyl chitosan has been reported

386

MODIFIED NATURAL POLYSACCHARIDES AS NANOPARTICULATE DRUG DELIVERY DEVICES

(a)

(b)

FIGURE 10.34 (a) OVA-challenged allergic mice showing epithelial damage, luminal narrowing due to airway wall edema and obstruction with excess mucus, which is typical of bronchial inflammation. (b) Mice treated with theophylline-loaded thiolated chitosan showing considerable reduction in pulmonary inflammation, decreased epithelial damage, reduced goblet cell hyperplasia, and fewer infiltrating inflammatory cells in the interstitial and peribronchovascular regions compared to the other groups. (Reproduced from Ref. [109]).

to have the best blood compatibility in comparison with other N-acylchitosans (Npropionyl, N-butyryl, and N-pentanoyl chitosan) [114]. The nanoparticles of hexanoyl chitosan were prepared through ionotropic gelation with tripolyphosphate (TPP). They exhibited a spherical shape with a mean diameter of 324 nm. At 0.2, 0.4, and 0.6 mg/mL bovine serum albumin initial concentration, the encapsulation efficiency and loading capacity of hexanoyl-chitosan-TPP nanoparticles were 58.2, 44.5, and 28.1% and 14.1, 23.4, and 30.3%, respectively. The potential of polyethylene glycol-grafted chitosan (PEG-g-chitosan) (Fig. 10.35) nanoparticles as a system for improving the systemic absorption of insulin following nasal administration has been investigated [115]. The addition of PEG was also considered in order to improve the biocompatibility of chitosan [116]. The synthetic pathway of PEG-g-chitosan was first reported by Harris et al. [117].

OH O O

HO NH

n

O O m

FIGURE 10.35 PEG-g-chitosan.

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The insulin-loaded nanoparticles (size = 150–300 nm, loading efficiency 20–39%) were prepared by the ionotropic gelation of PEG-g-chitosan solution using tripolyphosphate ions as the crosslinking agent. The nanoparticle formed a compact core surrounded by a fluffy coat of PEG. Sustained release was observed, and the mass of chitosan used had an influence on the diffusion of insulin as well as the rate of degradation of the nanoparticles. Higher mass lead to slower release. The rate of insulin release was also affected by the PEG content; increasing the PEG content increased the percentage release. The plasma glucose level of rabbits was monitored after intranasal administration. The glucose level was reduced at 54 and 68% of the basal level at 1 and 4 h after administration, respectively. The control insulin solution did not reduce blood glucose level significantly (< 20% decrease). 10.7 CONCLUSIONS AND FUTURE TRENDS Polysaccharides produced from renewable resources have been extensively studied for controlled drug release. Presently, the trend is to improve the performance of these polysaccharides by modifying them with synthetic biocompatible polymers or molecules. Nanoparticles composed of biocompatible materials have been used to increase the aqueous solubility of hydrophobic drugs via solubilization within the hydrophobic core of the nanoparticles. This is a promising approach to solubilizing drugs and eliminating the use of excipients such as Cremophor EL, which has been shown to cause hypersensitivity reactions. These modifications are expected to increase: encapsulation/loading efficiency, mucoadhesiveness, targeting properties, biocompatibility, internalization, performance of drugs, and also patient compliance in the case of infectious diseases such as tuberculosis (TB). For instance, quitting the TB treatment program leads to the especially dangerous multidrug-resistant form of TB, and over 400,000 new cases of multidrug-resistant TB are diagnosed every year. It is important to note that no new drugs have entered the market in the last 50 years; optimization of the delivery systems for the current drugs is the solution. Infectious diseases such as tuberculosis, malaria, and HIV/AIDS have a very high incidence in developing countries. Enormous amount of funding is being injected in research in that field from private/ public organizations such as WHO (US$ 5.67 million) and The Bill & Melinda Gates Foundation (US$ 350 million) [117]. Modified polysaccharides having shown their significant potential in the field of drug delivery can provide potential solutions to effective targeting of diseases affecting both developed and developing countries [118]. REFERENCES 1. Robinson, J. R., and Lee, H. L. (Eds.) (1987). Drugs and the Pharmaceutical Science, Controlled Drug Delivery Fundamentals and Applications, vol. 29. New York: Marcel Dekker. 2. Chasin, M., and Langer R. (Eds.) (1990). Drugs and the Pharmaceutical Science, Biodegrabable Polymers as Drug Delivery Systems, vol. 45. New York: Marcel Dekker.

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INDEX

2,2 -azobisisobutyronitrile (AIBN), 6 A␤-peptide, 185 Administration Nasal, 385 Oral, 379 Agglutination, 161 Aggregation behavior, 156 Alginate, 338, 341 Alpha carbon, 168 Alzheimer disease, 185 Amines, 156 Primary, 156 Quaternary, 159 Secondary, 159 Tertiary, 159 Amylopectin, 194, 195 Anticoagulant activity, 161 Antitumour effect, 146 Arachis hypogaea, 292 Asialoglycoprotein receptors, 120, 263, 324 Asthma, 385 Atomic force microscopy, 156, 344, 363

Bacillus anthracis, 208 Bacterial toxins, 171, 172 Barbituratic acid, 4 Bioconjugates, 143, 167, 168 Nucleic acid glycopolymer, 175 Oligodinucleotides glycopolymers, 178, 179 Protein glycopolymer, 174, 313 Bioconjugation, 33, 46, 62, 167 Random, 171 Site specific, 172 Biohybrids, 168 Biotin, 33, 48, 49, 69, 70, 180, 181 Boltron polyester, 265, 268 Bovine serum albumin, 172, 176, 177, 313, 337 Breast cancer, 291 Carbon nanotubes, 45, 190, 312 Closed ended, 192 Multi walled, 190, 197, 199, 200, 202, 203 Open ended, 192

Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels, Edited by Ravin Narain C 2011 John Wiley & Sons, Inc. Copyright

397

398

INDEX

Carbon nanotubes (Continued ) Pristine, 192 Shortened, 192 Single walled, 190, 191, 194, 203, 208 Carboxymethyl chitin, 367 Carrageenan, 360 Cavavalia ensformis, 282 Cellular uptake, 144 Cellulose, 361 Cellulose-g-PDMAEMA, 363, 364 Cellulose-g-poly(N, N-dimethylacrylamide), 363 Ethylcellulose-g-polyacrylic acid, 364 Hydroxypropyl cellulose-g-polyethyleneoxide, 384 Modified cellulose, 384 Cellulose acetate, 203 Chain end-functionality, 151 Chain transfer agent, 153, 154 Charge density, 146, 156, 157, 158 Characterization Contact angle, 322 Ellipsometry, 321 Microscopic, 320 Spectroscopic, 318 Thermogravimetric, 322 Chelate effect, 263 Chitosan, 359 Carboxymethyl chitosan, 367 Chitosan-g-stearic acid, 369 Chondroitin sulphate-g-polycaprolactone, 368 Lactic acid-g-chitosan, 363 Modified chitosan, 380 Polycaprolactone-g-chitosan, 368 Polyethylene oxide-g-chitosan, 363 Polylactide-g-chondroitin sulphate, 363 Thiolated chitosan, 363, 381, 385 Cholera toxin, 289 Chondroitin Sulfate, 185 Click chemistry, 271, 310 Cu(I), 5, 46, 66, 101 Thio-ene, 102 Click cycloaddition, 271 Cluster glycoside effect, 120, 145, 308, 325 Cobalt nanoparticles, 234 Coil-helix transition, 134 Complex formation, 158, 159

Concanavalin A, 6, 18, 19, 40, 42, 43, 46, 47, 50, 66, 82, 96, 282–284 Convergent synthesis, 266–267 Co-polymers Diblock, 27, 28, 40, 42, 48, 62–68, 73, 80–81, 97 Random, 6, 18, 27, 28, 33, 70, 81, 100, 101, 102 Star, 28, 41, 42, 43, 65 Triblock, 27, 28, 41, 42, 43 Core shell micelles, 134 Cross-linked iron oxide nanoparticles, 231 Cross-linked micelles, 67 Crew cut micelles, 378 Cyclodextrin, 43, 65, 146, 184, 186, 276, 311 Cytotoxicity, 145, 155 DC SIGN receptors, 225, 238, 242, 243, 244 De Gennes dense packing, 263 Dendrimers, 262–263 Carbohydrate based, 277 Carbohydrate centered, 274–276 Carbohydrate coated, 268–274 Carbosilane, 265, 268, 271, 276 Convergent approach, 265–268 Divergent approach, 265–266 Fluorescent, 273 Gallic acid dendrimer, 268, 271, 286 Polyphenylene dendrimer, 271 Dendrons, 266 Dicumyl peroxide, 26 DNA unpackaging, 145 Doxorubicin, 368 Drug delivery, 186 Dilution effect, 276 E and P selectins, 234, 244, 245 Electrospinning, 342 End Functionalization, 46, 73, 74, 82, 99 Endocytosis, 239 Enhanced permeability and retention, 357 Enzymatic, 33, 62, 63 Enzymatic trans-esterification, 4 Erythrina cristagalli, 282 Escherichia coli, 66, 287 Extracellular matrix, 337 Extravasation, 155

INDEX

Fibroblast growth factor, 2 (FGF-2), 29, 33 Fischer glycoside synthesis, 4 Fluorescence inhibition assay, 156 Fluorescent gold glyconanoparticles, 244 Fluorescent probes, 230 Folic acid, 367 Functionalization techniques Grafting from, 314 Grafting to, 313 Langmuir Blodgett, 315 Layer by layer, 312 Self-assembled monolayer, 315 FimH, 287 Galactose, 291 Galanthus nivalus, 282 Gel electrophoresis, 156, 157 Gene delivery, 155 Non-viral, 143, 144, 155 Viral, 143 Glucose oxidase (Gox), 206 Glycan binding epitopes, 245 Glycoblotting, 223 Glycocalyx, 119, 120, 239 Glycoconjugates, 145, 215, 223, 237 Glycodendrimers, 261 Glycolipids, 215, 227, 245 Glyconanoparticles, 214 Glycopeptide, 173 Glycopolymers, 147 Biotin Functionalized, 170 Cationic, 143 Hyperbranched, 43 Natural, 120, 357, 358 pH-sensitive, 131 Protected, 125 Synthetic, 198 Telechelic, 176, 180 Temperature sensitive, 135 Unprotected, 125 Glycoproteins, 215, 227 Glycosamnioglycans, 178, 185, 337 Glycosphingolipids, 216, 218, 219, 223, 241, 242 Glycosurfaces, 307 Gold, 46, 50, 69 Gold-iron glyconanoparticles, 231, 232, 233 Gold nanoparticles, 69 Gold nanoparticles synthesis, 215, 216

399

Brust method, 216 Monolayer protected clusters, 216 Non-covalent stabilization, 217 Turkevich method, 216 Grignard reactions, 3, 18 HABA, 181 Hemaglutinin, 181 Heparin sulfate, 185 HIV, 357 Hybrid nanoclusters, 223 Hydrodynamic diameter, 136, 137 Hydrogels Biomimetic, 348 Carbohydrate derived, 338 Chemically cross-linked, 338 Injectable, 347 Photo cross-linked, 341 Physically cross-linked, 338 Self aggregating, 343 Immunogenic peptide, 223 Initiator, 148, 149, 150, 153, 154 Interactions Carbohydrate-carbohydrate, 215, 218, 219, 220, 223 Carbohydrate-protein, 171, 179, 183, 185, 214, 215, 223, 227, 245 Multivalent, 230, 263 Non-covalent interactions, 236 Non-specific, 223, 245 Protein-protein, 179, 219, 229, 245 Lectins, 263 Agglutinin RCA120 18, 19, 26, 46, 50, 70, 100, 227, 326 BSl-B4, 19 Castor bean lectin, 6 Concavanilin A, 6, 40, 227, 229 Mannose binding lectin, 176, 177 Peanut lectin, 18, 69 Ricinus communis agglutinin, 18 Wheat germ agglutinin, 19, 74, 80 Lewis X antigen, 218, 234, 236, 244 Limax flavus, 282, 285 Major histocompatibility complex class II, 238 Malaria, 357

400

INDEX

Maltose-rotaxane conjugates, 184 Membrane, 46 Methyl D-glucopyranoside, 282 Methyl D-mannopyranoside, 282, 288, 289 Micelles, 28, 42, 43, 66–67, 102 Microgels, 337 Modification of nanotubes, 192 Ball milling method, 202 Covalent, 193, 196, 202, 204 Grafting from, 196, 197 Grafting to, 196, 197 Hybrid, 193, 204 Layer by layer deposition, 201 Non-covalent, 193, 195, 204 Oxidation, 192 Molecular recognition, 180 Molecular weight, 146–149, 153–156, 158–160, 162 Molecular weight distribution, 148, 158–160 Monomers Acrylamide, 6 Methacrylate, 6 Other vinyl containing, 19 Styrene based, 18 Mucins, 178 Multivalent effect, 263–265 N-acetyl neuraminic acid, 161 Nanocage, 68 Nanoparticles, 69, 357 Nanotechnology, 189, 190, 214, 240 Neoglycoconjugates, 220, 232, 236 N/P ratio, 159, 160 Nuclear translocation, 155 Octopus glycosides, 274 Oligomannoside, 278 Oligosaccharides, 169, 175, 183, 371 Ovalbumin, 385 Oxime linkage, 178 Pathogen associated molecular patterns, 184 Pathogen recognition, 183 PEGylation, 145 P-glycoprotein, 383 Phagocytosis, 144

Poly(amido amine), 265, 268, 269, 274, 282–284, 286, 287, 289, 292 Poly(ε-caprolactone), 199, 200, 375 Polyethylenimine, 146, 147, 156 Poly(lactide-co-glycolide) (PLGA), 382 Poly-L-Lysine, 147, 156, 160 Polymerization Anionic chain, 2, 74 Atom transfer radical, 2, 33, 123, 152, 173, 196, 197, 180, 315, 362, 363 Cationic chain, 2, 48, 80 Controlled/living radical, 20 Conventional, 122 Cyanoxyl-mediated, 2, 28, 180, 185 Emulsion, 342 Free radical, 5 Nitroxide-mediated, 2, 20, 149, 170 Reversible addition fragmentation chain transfer, 2, 50, 127, 151, 153, 154, 172, 178, 196, 199, 201, 218, 315, 363 Ring-opening, 2, 71, 82, 170, 362 Ring opening metathesis, 82 Surface-initiated, 314 TEMPO-mediated, 2 Ultraviolet, 342 Poly( p-phenylene ethynylene), 289 Polyplexes, 144, 145, 156 Poly(propylene imine), 265, 268 Polysaccharides Agarose, 358 Amylopectin, 361 Amylose, 361, 377 Carrageenan, 360 Cellulose, 362, 384 Chitosan, 146, 160, 194, 201, 202, 204, 238, 239, 338, 380 Dextran, 146, 231, 233, 244, 338, 372 Heparin, 29, 33, 161, 183, 324 Hyaluronan, 377 Hydroxy ethylcellulose, 364 Schizophyllan, 146, 194 Starch, 361 Polythiophene, 162, 183 Polyvalency, 161, 215 Postfunctionalization, 97 Amide linkage, 98 Click approach, 101 Other non-click approach, 103 Postmodification, 172

INDEX

Precipitation assay, 282 Pro-angiogenic protein growth factor, 185 Proteoglycans, 218 Proximity/statistical effect, 264 Pullulan, 343 Pullulan acetate, 366 Pyrene, 204 Quantum dots Glyco-coated, 230, 234–239 PEG coated, 238 TOPO coated, 238 Reductive amination, 269 Reticuloendothelial system (RES), 231 Ricin, 182 Rods, 40, 42, 66 Saturosporine, 144 Selectins, 96, 183, 184 Self assembly, 42, 62, 65–68 Self organizing nanogels, 343 Semiconductor nanocrystals, 234, 235 Shigatoxin-1, 19 Sialic acid, 181, 285 Sialidase, 287 Silica coated iron nanoparticles, 234, 240 siRNA, 49, 174 Solid-phase binding assay, 282, 291, 293 Stars, 65

401

Streptavidin, 180, 181, 314 Sugars Amino, 4 Halogen, 3 Isopropyl-idene, 3 Surface grafting, 44–46, 68–71 Surface and particle modification, 68 Surface plasmon resonance, 177, 180, 182, 223, 227, 229, 244, 291, 327 Symbiosis, 214 T-antigen, 291–292 TEMPO, 21 Thomsen-Friedenreich, 291 Toll like receptors, 184 Transfection, 143, 145–147, 155–159 Polymer based, 157 Viral based, 143 Trehalose, 276 Tris(hydroxymethyl)aminomethane, 271 Tuberculosis, 357 Valency, 263 Vesicles, 40–43, 66 Vibrio cholerae, 289 Vicia villosa, 282 Viscoelastic properties, 345 Xenopus Oocytes, 157, 159 Zeta potential, 156

FIGURE 2.3 (See text for details.)

FIGURE 4.3 (See text for details.)

Intensity (a.u.)

25000 20000 15000 10000 5000 0 0

Electronic microscopy

Raman spectrum

0 0

0

0 Intensity

Structure

300 600 900 1200 1500 1800 2100 2400 2700 3000 cm Raman Spectrum of 90% SWNT

0

0

0 0 0 0

FIGURE 5.1 (See text for details.)

0

0

0

0 0 0 0 0 Wavenumber (cm)

0

0

0

O

OH O

O

EDC / NHS

O

pH 6, r. t

N

HO O

OH

O

O O

O O

SWNT-COOH

O O

O

O

N

N

O

P(APMA-b-LAEMA)

SWNT-NHS (1) P(APMA-b-LAEMA), pH 9, r. t

(2) pH 6

Sugar groups DNA Amino groups

Complex with DNA

Surface-functionalized SWNT with polymer and DNA

Surface-functionalized SWNT with polymer

SCHEME 5.7 (See text for details.)

FIGURE 6.1 (See text for details.)

HO HO O O HO

HO OH

HO

O OH O HO

HO OH

OH HO

HO O

O O

O

S

OH

O O HO

S S

O O HO

S

S

O O

S O HO

HO

HO

O

O

HO OH O O OH HO

O

O

OH

O HO OH HO OH O HO

HO OH OH

HO HO O O

HO OH

HO

S

O O OH

HO OH O O

OH

S

S

OH

OH OH

HO S

S

HO

O OH HO O HO

O

S O

HO

HO HO

OH HO

O O HO

OH

OH HO O O HO

OH

OO

S

O OH HO OH O HO O O O

OH

OH

O O

HO

OH OH O HO HO O HO

OH

HO

HO

O

HO

OH

OH O HO O HO

OH OH

OH O OH

O OH HO OH

OH

OH OH

(a)

(b)

FIGURE 6.2 (See text for details.)

(a)

(b)

FIGURE 6.3 (See text for details.)

(c)

(ii) ozonolysis

(i) extraction

cell HO HO

OH HOOC O O

HO AcHN

O

OH

OH

O

O

O OH HO

OH HOOC O O

HO

OH

O OH

Cr Cr

HO

HO

OC

H Ac O HN

HO AcHN

OH

OH

Cr

HO

O OH

MS-based structural analysis Cr

HO

HN

O

OH

OH O O OH HO

O OH

HN

O O

O

OH

OH

OH

(iii) GSLs-blotting

O OH O HO

OH

S

O OH O

Au N

O

S m/z

Au

SPR-based functional analysis

GSLs-GNP

FIGURE 6.5 (See text for details.)

aoGNP

Virus Cell adhesion

Glycoprotein half-life

Antibodies

Bacterial toxin

Bacteria (a) Types of N-glycans

α2 α2

α2 α3

α3

α2 α6

α6

α6

α6

β4

β4

β4

β2

β2

β2

α3

α6

α6

α3

α3

α6

β4

β4

β4

β4

β4 α6

β4

β

β

Asn

β

Asn

Oligomannose

Asn

Complex

Mannose

Galactose

Fucose

α6

Hybrid N-acetyl-glucosamine

N-acetyl-neuraminic acid (b)

FIGURE 6.8 (See text for details.)

OH OH

OH O

OH O

O

NH

HO OH

O

O

O

Au

S

6

n

(a) Targets

Targets

PCB electrodes PNA

SNA

SNA

PNA

SNA

immobilized

immobilized

Gold Nickel Copper Fiberglass

asialofetuin TF-AuNP

fetuin

MHDA

(b)

FIGURE 6.9 (See text for details.)

(b)

(a) (c)

FIGURE 6.12 (See text for details.)

gp 120-QD

PNA

HLA-DR

FIGURE 6.14 (See text for details.)

O

O

O OH O

HO HO

R= HO

O

O

HO HO

O

2

O

OH

4

3 OH O

HO HO

OH O

OH O HO

OH O

OH O HO

OH O

HO HO

OH O

HO HO

OH O

OH

1

SH

OH O

OH O OH O

OH O HO

O

HO HO O

OH

5 OH O

HO HO

OH O

OHO HO

6 OH O

O HO

OH

OH O

O HO

OH

O

OH

O

OH

7

200

(a) H O O

O

O HO

H

O

H

O

O

C O O H

D

S

S

S

C

S S S

S O

S

Q 5–

S

se

D

D

Q

Q

6–

HOOC

e

o tri

os

to

C

a

D

4–

Q

O

M

se

1–

to al

MPA–QD

n Pa

0

M

S CdTe

G PE

O

O

O

S

SH HO

COOH

50

S

S

O

S

S H

H

O O

C

O

HO

COOH

HO

100

O

O

S CdTe

HOOC

(d)

150

H

O

H

HO

Fluorescent intens ty in nucleus

O R

Sugar–QD

(b)

(c)

FIGURE 6.15 (See text for details.)

(b)

(a)

(c)

FIGURE 6.16 (See text for details.)

Glc-GNP (90 µm)

Lac-GNP (90 µm)

x8

B16F10

GNP suspension x 80

5 min, 37 °C

(b) Control B16F10

GNP-treated B16F10

B16F10 (105 cells/animal)

1000 rpm r.t

mock Glc-GNP Lac-GNP

mock 3 weeks 1 week

Glc-GNP (90 µm)

Cell viability Lac-GNP (90 µm)

Lung tumoral foci score/ Anatomopathologic studies

(a)

(c)

FIGURE 6.17 (See text for details.)

OH OH

HO

CO2H O

O

AcHN

OH

HO

O OH

HO

O O O

OH O

S NHAc

A

B

C

D

E

F

N

OH

OH HO

H2N

H2N H2N

H2N H2N NH2

HO

OH OH

CO2H O

AcHN HO

O HO

NH2 NH2 NH2

OH O OH

O O O

OH HO

OH

OH O

S NHAc

NH N H

χ105

GNP-sLex

FIGURE 6.19 (See text for details.)

No B16F10

Engineered Carbohydrate-Based Materials for Biomedical Applications: Polymers, Surfaces, Dendrimers, Nanoparticles, and Hydrogels